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<h1 class="advertising-feature"><strong>Premium RRBS technology: cost-effective DNA methylation mapping with superior coverage</strong></h1>
<ul class="citation dates">
<li style="text-align: left;" class="vcard last-author"><a href="#auth-5" class="name"><span class="fn">Christoph Bock</span></a><sup><a href="#a2">2</a></sup><sup href="#affil-auth">, </sup></li>
<li style="text-align: left;" class="vcard"><a href="#auth-4" class="name"><span class="fn">Sharon Squazzo</span></a><sup><a href="#a3">3</a></sup><sup>, </sup></li>
<li style="text-align: left;" class="vcard"><a href="#auth-3" class="name"><span class="fn">Miklos Laczik</span></a><sup><a href="#a1">1</a></sup><sup>, </sup></li>
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<p>Published online <time datetime="2016-01-28">28 January 2016 </time></p>
<p></p>
<p><a href="http://www.nature.com/nmeth/journal/v13/n2/full/nmeth.f.391.html" class="alert button"><span style="font-weight: 400;">CHECK OUT THE NATURE METHODS PAPER</span></a></p>',
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<li><strong>Cost-efficient</strong> – Multiplex up to 6 samples/sequencing lane</li>
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<div class="small-6 columns"><br />
<p><b>Superior coverage</b></p>
<p>Comparison of CpG coverage between competing technologies.</p>
<p><strong><em><small>They love it! </small></em></strong><br /><em><small>The new Diagenode Premium RRBS Kit makes it easy to use RRBS cost-effectively and with high throughput, using early sample pooling and multiplex sequencing. Most importantly, the method provides an improved coverage of up to 4 million CpGs for the human genome. We successfully used this protocol on more than 1,000 samples comprising of six different species, various cancers, FFPE and lowinput samples. <br /><strong>Paul Datlinger and Christoph Bock, </strong><strong>CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria </strong></small></em></p>
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<div class="small-6 columns">
<p><b>Accurate determination of DNA methylation level</b></p>
Excellent results were obtained using Diagenode’s Premium RRBS kit: almost 90% alignment rate, 4.1 million CpGs covered and bisulfite conversion rates around 99.5% for all samples. DNA methylation percentages in the region of IGF2 obtained with the Diagenode’s Premium RRBS Kit. Two human cell lines were analyzed: Gm12878 and MCF7. The MCF7 cell line was studied in duplicates. Each peak represents the DNA methylation percentage at one CpG. The methylated CpGs are shown in red and the unmethylated CpGs in grey.
<p><br /><br /></p>
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<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/landing-pages/rrbs_how_it_works.jpg" alt="rrbs how it works" style="display: block; margin-left: auto; margin-right: auto;" /><br /><br /></p>
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<div class="small-6 columns">
<p><b>How it works</b></p>
By cutting the genome using the <strong>restriction enzyme MspI</strong> (CCGG target sites) followed by size selection, DNA is enriched to represent <strong>CpG-rich genomic regions</strong> (including CpG islands, CpG island shores, enhancers, and other gene-regulatory elements), which are particularly relevant for epigenetic regulation. Similar to exome-sequencing for mutation discovery, the RRBS protocol enriches for some of the most interesting target regions and thereby achieves a reduction in sequencing cost of a factor of 10-20 compared to whole genome bisulfite sequencing.</div>
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<table width="1039">
<tbody>
<tr></tr>
<tr>
<td width="281"> </td>
<td width="379">
<h4>Premium RRBS Kit</h4>
</td>
<td width="379">
<h5>Illumina® RRBS protocol</h5>
</td>
</tr>
<tr>
<td><strong>DNA input</strong></td>
<td>100 ng</td>
<td>2-5 µg</td>
</tr>
<tr>
<td><strong>Multiplexing</strong></td>
<td>Pooling of 6 samples (one HiSeq lane)</td>
<td>no guidelines</td>
</tr>
<tr>
<td><strong>Bisulfite conversion</strong></td>
<td>One bisulfite reaction per pool (6 samples)</td>
<td>One bisulfite reaction per sample</td>
</tr>
<tr>
<td><strong># purification steps</strong></td>
<td>2</td>
<td>7</td>
</tr>
<tr>
<td><strong>Key features</strong></td>
<td width="379">low input - cost-effective - optimized for high-throughput - complete kit</td>
<td width="379">high input - not optimized for high-throughput - reagents from different providers</td>
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<h1 class="advertising-feature"><strong>Premium RRBS technology: cost-effective DNA methylation mapping with superior coverage</strong></h1>
<ul class="citation dates">
<li style="text-align: left;" class="vcard last-author"><a href="#auth-5" class="name"><span class="fn">Christoph Bock</span></a><sup><a href="#a2">2</a></sup><sup href="#affil-auth">, </sup></li>
<li style="text-align: left;" class="vcard"><a href="#auth-4" class="name"><span class="fn">Sharon Squazzo</span></a><sup><a href="#a3">3</a></sup><sup>, </sup></li>
<li style="text-align: left;" class="vcard"><a href="#auth-3" class="name"><span class="fn">Miklos Laczik</span></a><sup><a href="#a1">1</a></sup><sup>, </sup></li>
<li style="text-align: left;" class="vcard"><a href="#auth-2" class="name"><span class="fn">Paul Datlinger</span></a><sup><a href="#a2">2</a></sup><sup>, </sup></li>
<li style="text-align: left;" class="vcard c1"><a href="#auth-1" class="name"><span class="fn">Anne-Clémence Veillard</span></a><sup><a href="#a1">1</a></sup><sup>, </sup></li>
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<p>Nature Methods 13 (2016)</p>
<p>Published online <time datetime="2016-01-28">28 January 2016 </time></p>
<p></p>
<p><a href="http://www.nature.com/nmeth/journal/v13/n2/full/nmeth.f.391.html" class="alert button"><span style="font-weight: 400;">CHECK OUT THE NATURE METHODS PAPER</span></a></p>
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<p> </p>
<div class="small-12 medium-4 large-4 columns"><center></center><center></center><center></center><center><a href="https://www.ncbi.nlm.nih.gov/pubmed/30429608" target="_blank"><img src="https://www.diagenode.com/img/banners/banner-nature-publication-580.png" alt="Click here to read more about MeDIP " caption="false" width="80%" /></a></center></div>
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<h3 style="text-align: justify;">Sensitive tumour detection and classification using plasma cell-free DNA methylomes<br /><a href="https://www.ncbi.nlm.nih.gov/pubmed/30429608" target="_blank">Read the publication</a></h3>
<h3 class="c-article-title u-h1" data-test="article-title" itemprop="name headline" style="text-align: justify;">Preparation of cfMeDIP-seq libraries for methylome profiling of plasma cell-free DNA<br /><a href="https://www.nature.com/articles/s41596-019-0202-2" target="_blank" title="cfMeDIP-seq Nature Method">Read the method</a></h3>
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<p>Perform <strong>MeDIP</strong> (<strong>Me</strong>thylated <strong>D</strong>NA <strong>I</strong>mmuno<strong>p</strong>recipitation) followed by qPCR or NGS to estimate DNA methylation status of your sample using a highly sensitive 5-methylcytosine antibody. Our MagMeDIP kit contains high quality reagents to get the highest enrichment of methylated DNA with an optimized user-friendly protocol.</p>
</div>
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<h3><span>Features</span></h3>
<ul>
<li>Starting DNA amount: <strong>10 ng – 1 µg</strong></li>
<li>Content: <strong>all reagents included</strong> for DNA extraction, immunoprecipitation (including the 5-mC antibody, spike-in controls and their corresponding qPCR primer pairs) as well as DNA isolation after IP.</li>
<li>Application: <strong>qPCR</strong> and <strong>NGS</strong></li>
<li>Robust method, <strong>superior enrichment</strong>, and easy-to-use protocol</li>
<li><strong>High reproducibility</strong> between replicates and repetitive experiments</li>
<li>Compatible with <strong>all species </strong></li>
</ul>',
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<h3><span>How it works</span></h3>
<p>In brief, after the cell collection and lysis, the genomic DNA is extracted, sheared, and then denatured. In the next step the antibody directed against 5 methylcytosine and antibody binding beads are used for immunoselection and immunoprecipitation of methylated DNA fragments. Then, the IP’d methylated DNA is isolated and can be used for any subsequent analysis as qPCR, amplification, hybridization on microarrays or next generation sequencing.</p>
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'info2' => '<p>The kit MagMeDIP contains all reagents necessary for a complete MeDIP-qPCR workflow. Two MagMeDIP protocols have been validated: for manual processing as well as for automated processing, using the Diagenode’s IP-Star Compact Automated System (please refer to the kit manual).</p>
<ul>
<li><strong>Complete kit</strong> including DNA extraction module, IP antibody and reagents, DNA isolation buffer</li>
<li><strong>Quality control of the IP:</strong> due to methylated and unmethylated DNA spike-in controls and their associated qPCR primers</li>
<li><strong>Easy to use</strong> with user-friendly magnetic beads and rack</li>
<li><strong>Highly validated protocol</strong></li>
<li>Automated protocol supplied</li>
</ul>
<center><img src="https://www.diagenode.com/img/product/kits/fig1-magmedipkit.png" width="85%" alt="Methylated DNA Immunoprecipitation" caption="false" /></center>
<p style="font-size: 0.9em;"><em><strong>Figure 1.</strong> Immunoprecipitation results obtained with Diagenode MagMeDIP Kit</em></p>
<p style="font-size: 0.9em;">MeDIP assays were performed manually using 1 µg or 50 ng gDNA from blood cells with the MagMeDIP kit (Diagenode). The IP was performed with the Methylated and Unmethylated spike-in controls included in the kit, together with the human DNA samples. The DNA was isolated/purified using DIB. Afterwards, qPCR was performed using the primer pairs included in this kit.</p>
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'info3' => '<p>For DNA methylation analysis on the whole genome, MagMeDIP kit can be coupled with Next-Generation Sequencing. To perform MeDIP-sequencing we recommend the following strategy:</p>
<ul style="list-style-type: circle;">
<li>Choose a library preparation solution which is compatible with the starting amount of DNA you are planning to use (from 10 ng to 1 μg). It can be a home-made solution or a commercial one.</li>
<li>Choose the indexing system that fits your needs considering the following features:</li>
<ul>
<ul>
<ul>
<li>Single-indexing, combinatorial dual-indexing or unique dual-indexing</li>
<li>Number of barcodes</li>
<li>Full-length adaptors containing the barcodes or barcoding at the final amplification step</li>
<li>Presence / absence of Unique Molecular Identifiers (for PCR duplicates removal)</li>
</ul>
</ul>
</ul>
<li>Standard library preparation protocols are compatible with double-stranded DNA only, therefore the first steps of the library preparation (end repair, A-tailing, adaptor ligation and clean-up) will have to be performed on sheared DNA, before the IP.</li>
</ul>
<p style="padding-left: 30px;"><strong>CAUTION:</strong> As the immunoprecipitation step occurs at the middle of the library preparation workflow, single-tube solutions for library preparation are usually not compatible with MeDIP-sequencing.</p>
<ul style="list-style-type: circle;">
<li>For DNA isolation after the IP, we recommend using the <a href="https://www.diagenode.com/en/p/ipure-kit-v2-x24" title="IPure kit v2">IPure kit v2</a> (available separately, Cat. No. C03010014) instead of DNA isolation Buffer.</li>
</ul>
<ul style="list-style-type: circle;">
<li>Perform library amplification after the DNA isolation following the standard protocol of the chosen library preparation solution.</li>
</ul>
<h3><span>MeDIP-seq workflow</span></h3>
<center><img src="https://www.diagenode.com/img/product/kits/MeDIP-seq-workflow.png" width="110%" alt="MagMeDIP qPCR Kit x10 workflow" caption="false" /></center>
<h3><span>Example of results</span></h3>
<center><img src="https://www.diagenode.com/img/product/kits/medip-specificity.png" alt="MagMeDIP qPCR Kit Result" caption="false" width="951" height="488" /></center>
<p></p>
<p style="font-size: 0.9em;"><strong>Figure 1. qPCR analysis of external spike-in DNA controls (methylated and unmethylated) after IP.</strong> Samples were prepared using 1μg – 100ng -10ng sheared human gDNA with the MagMeDIP kit (Diagenode) and a commercially available library prep kit. DNA isolation after IP has been performed with IPure kit V2 (Diagenode).</p>
<p></p>
<p></p>
<center><img src="https://www.diagenode.com/img/product/kits/medip-saturation-analysis.png" alt=" MagMeDIP kit " caption="false" width="951" height="461" /></center>
<p></p>
<p style="font-size: 0.9em;"><strong>Figure 2. Saturation analysis.</strong> Clean reads were aligned to the human genome (hg19) using Burrows-Wheeler aligner (BWA) algorithm after which duplicated and unmapped reads were removed resulting in a mapping efficiency >98% for all samples. Quality and validity check of the mapped MeDIP-seq data was performed using MEDIPS R package. Saturation plots show that all sets of reads have sufficient complexity and depth to saturate the coverage profile of the reference genome and that this is reproducible between replicates and repetitive experiments (data shown for 50 ng gDNA input: left panel = replicate a, right panel = replicate b).</p>
<p></p>
<p></p>
<center><img src="https://www.diagenode.com/img/product/kits/medip-libraries-prep.png" alt="MagMeDIP x10 " caption="false" width="951" height="708" /></center>
<p></p>
<p style="font-size: 0.9em;"><strong>Figure 3. Sequencing profiles of MeDIP-seq libraries prepared from different starting amounts of sheared gDNA on the positive and negative methylated control regions.</strong> MeDIP-seq libraries were prepared from decreasing starting amounts of gDNA (1 μg (green), 50 ng (red), and 10ng (blue)) originating from human blood with the MagMeDIP kit (Diagenode) and a commercially available library prep kit. DNA isolation after IP has been performed with IPure kit V2 (Diagenode). IP and corresponding INPUT samples were sequenced on Illumina NovaSeq SP with 2x50 PE reads. The reads were mapped to the human genome (hg19) with bwa and the alignments were loaded into IGV (the tracks use an identical scale). The top IGV figure shows the TSH2B (also known as H2BC1) gene (marked by blue boxes in the bottom track) and its surroundings. The TSH2B gene is coding for a histone variant that does not occur in blood cells, and it is known to be silenced by methylation. Accordingly, we see a high coverage in the vicinity of this gene. The bottom IGV figure shows the GADPH locus (marked by blue boxes in the bottom track) and its surroundings. The GADPH gene is a highly active transcription region and should not be methylated, resulting in no reads accumulation following MeDIP-seq experiment.</p>
<p></p>
<ul>
<ul>
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'meta_description' => 'Perform Methylated DNA Immunoprecipitation (MeDIP) to estimate DNA methylation status of your sample using highly specific 5-mC antibody. This kit allows the preparation of cfMeDIP-seq libraries.',
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'description' => '<p><a href="https://www.diagenode.com/files/products/kits/premium-RRBS-spike-in%20controls-08_18.pdf"><img src="https://www.diagenode.com/img/buttons/bt-manual.png" /></a></p>
<p>The DNA methylation control package includes one methylated and one unmethylated spike-in controls together with their corresponding qPCR primer sets for assessing the efficiency of your non-plant MeDIP experiments (Methylated DNA Immunoprecipitation) carried out with Diagenode’s <a href="https://www.diagenode.com/en/p/magmedip-kit-x48-48-rxns">MagMeDIP qPCR Kit</a> and <a href="https://www.diagenode.com/en/p/auto-magmedip-kit-x48-48-rxns" target="_blank">Auto MagMeDIP qPCR Kit</a>.</p>
<p><span>Those spike-in controls are made from <em>A. thaliana</em><span>. Therefore, they can interfere with DNA samples derived from plant species.</span></span></p>
<p><em><strong>CAUTION</strong>: These spike-in controls are compatible with <span>Diagenode’s </span><a href="https://www.diagenode.com/en/p/magmedip-kit-x48-48-rxns">MagMeDIP qPCR Kit</a><span><span> </span>and<span> </span></span><a href="https://www.diagenode.com/en/p/auto-magmedip-kit-x48-48-rxns" target="_blank">Auto MagMeDIP qPCR Kit</a><span>. However</span>, those are not the ones directly provided in the kits. The spike-in controls provided in the kits are available separately with the <a href="https://www.diagenode.com/en/p/dna-methylation-control-package-V2-48-rxns">DNA methylation control package V2</a>.</em></p>',
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'meta_description' => 'DNA Methylation control package',
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'description' => '<div class="row">
<div class="large-12 columns">
<div style="text-align: justify;" class="small-12 medium-8 large-8 columns">
<h2>Complete solutions for DNA methylation studies</h2>
<p>Whether you are experienced or new to the field of DNA methylation, Diagenode has everything you need to make your assay as easy and convenient as possible while ensuring consistent data between samples and experiments. Diagenode offers sonication instruments, reagent kits, high quality antibodies, and high-throughput automation capability to address all of your specific DNA methylation analysis requirements.</p>
</div>
<div class="small-12 medium-4 large-4 columns text-center"><a href="../landing-pages/dna-methylation-grant-applications"><img src="https://www.diagenode.com/img/banners/banner-dna-grant.png" alt="" /></a></div>
<div style="text-align: justify;" class="small-12 medium-12 large-12 columns">
<p>DNA methylation was the first discovered epigenetic mark and is the most widely studied topic in epigenetics. <em>In vivo</em>, DNA is methylated following DNA replication and is involved in a number of biological processes including the regulation of imprinted genes, X chromosome inactivation. and tumor suppressor gene silencing in cancer cells. Methylation often occurs in cytosine-guanine rich regions of DNA (CpG islands), which are commonly upstream of promoter regions.</p>
</div>
<div class="small-12 medium-12 large-12 columns"><br /><br />
<ul class="accordion" data-accordion="">
<li class="accordion-navigation"><a href="#dnamethyl"><i class="fa fa-caret-right"></i> Learn more</a>
<div id="dnamethyl" class="content">5-methylcytosine (5-mC) has been known for a long time as the only modification of DNA for epigenetic regulation. In 2009, however, Kriaucionis discovered a second methylated cytosine, 5-hydroxymethylcytosine (5-hmC). The so-called 6th base, is generated by enzymatic conversion of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine by the TET family of oxygenases. Early reports suggested that 5-hmC may represent an intermediate of active demethylation in a new pathway which demethylates DNA, converting 5-mC to cytosine. Recent evidence fuel this hypothesis suggesting that further oxidation of the hydroxymethyl group leads to a formyl or carboxyl group followed by either deformylation or decarboxylation. The formyl and carboxyl groups of 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) could be enzymatically removed without excision of the base.
<p class="text-center"><img src="https://www.diagenode.com/img/categories/kits_dna/dna_methylation_variants.jpg" /></p>
</div>
</li>
</ul>
<br />
<h2>Main DNA methylation technologies</h2>
<p style="text-align: justify;">Overview of the <span style="font-weight: 400;">three main approaches for studying DNA methylation.</span></p>
<div class="row">
<ol>
<li style="font-weight: 400;"><span style="font-weight: 400;">Chemical modification with bisulfite – Bisulfite conversion</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Enrichment of methylated DNA (including MeDIP and MBD)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Treatment with methylation-sensitive or dependent restriction enzymes</span></li>
</ol>
<p><span style="font-weight: 400;"> </span></p>
<div class="row">
<table>
<thead>
<tr>
<th></th>
<th>Description</th>
<th width="350">Features</th>
</tr>
</thead>
<tbody>
<tr>
<td><strong>Bisulfite conversion</strong></td>
<td><span style="font-weight: 400;">Chemical conversion of unmethylated cytosine to uracil. Methylated cytosines are protected from this conversion allowing to determine DNA methylation at single nucleotide resolution.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Single nucleotide resolution</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Quantitative analysis - methylation rate (%)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Gold standard and well studied</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Compatible with automation</span></li>
</ul>
</td>
</tr>
<tr>
<td><b>Methylated DNA enrichment</b></td>
<td><span style="font-weight: 400;">(Hydroxy-)Methylated DNA is enriched by using specific antibodies (hMeDIP or MeDIP) or proteins (MBD) that specifically bind methylated CpG sites in fragmented genomic DNA.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Resolution depends on the fragment size of the enriched methylated DNA (300 bp)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Qualitative analysis</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Compatible with automation</span></li>
</ul>
</td>
</tr>
<tr>
<td><strong>Restriction enzyme-based digestion</strong></td>
<td><span style="font-weight: 400;">Use of (hydroxy)methylation-sensitive or (hydroxy)methylation-dependent restriction enzymes for DNA methylation analysis at specific sites.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Determination of methylation status is limited by the enzyme recognition site</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Easy to use</span></li>
</ul>
</td>
</tr>
</tbody>
</table>
</div>
</div>
<div class="row"></div>
</div>
</div>
<div class="large-12 columns"></div>
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'meta_description' => 'Complete, optimized solutions for analyzing DNA methylation manually or on our automated system.',
'meta_title' => 'DNA Methylation - Bisulfite sequencing - Epigenetics | Diagenode',
'modified' => '2019-03-25 10:07:27',
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<div style="text-align: justify;" class="large-12 columns">Bisulfite modification of DNA is the most commonly used, "<strong>gold standard</strong>" method for DNA methylation studies providing <strong>single nucleotide resolution</strong>. T<span style="font-weight: 400;">his technology is based on the chemical conversion of unmethylated cytosine to uracil. Methylated cytosines are protected from this conversion allowing to determine DNA methylation at the singe nucleotide level.</span></div>
<div style="text-align: justify;" class="large-12 columns"></div>
<div style="text-align: justify;" class="large-12 columns">Various analyses can be performed on the altered sequence to retrieve this information: bisulfite sequencing, pyrosequencing, methylation-specific PCR, high resolution melting curve analysis, microarray-based approaches, and next-generation sequencing.
<h3>How it works</h3>
Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected (see Figure 1).
<p class="text-center"><img src="https://www.diagenode.com/img/applications/bisulfite.png" /><br />Figure 1: Overview of bisulfite conversion of DNA</p>
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'meta_title' => 'Bisulfite sequencing(Bis-Seq) - Bisulfite conversion - DNA Methylation | Diagenode',
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<div class="small-12 medium-3 large-3 columns"><center><a href="https://www.ncbi.nlm.nih.gov/pubmed/30429608" target="_blank"><img src="https://www.diagenode.com/img/banners/banner-nature-publication-580.png" /></a></center></div>
<div class="small-12 medium-9 large-9 columns">
<h3>Sensitive tumour detection and classification using plasma cell-free DNA methylomes<br /><a href="https://www.ncbi.nlm.nih.gov/pubmed/30429608" target="_blank">Read the publication</a></h3>
<h3 class="c-article-title u-h1" data-test="article-title" itemprop="name headline">Preparation of cfMeDIP-seq libraries for methylome profiling of plasma cell-free DNA<br /><a href="https://www.nature.com/articles/s41596-019-0202-2" target="_blank" title="cfMeDIP-seq Nature Method">Read the method</a></h3>
</div>
</div>
<div class="row">
<div class="large-12 columns"><span>The Methylated DNA Immunoprecipitation is based on the affinity purification of methylated and hydroxymethylated DNA using, respectively, an antibody directed against 5-methylcytosine (5-mC) in the case of MeDIP or 5-hydroxymethylcytosine (5-hmC) in the case of hMeDIP.</span><br />
<h2></h2>
<h2>How it works</h2>
<p>In brief, Methyl DNA IP is performed as follows: Genomic DNA from cultured cells or tissues is prepared, sheared, and then denatured. Then, immunoselection and immunoprecipitation can take place using the antibody directed against 5 methylcytosine and antibody binding beads. After isolation and purification is performed, the IP’d methylated DNA is ready for any subsequent analysis as qPCR, amplification, hybridization on microarrays or next generation sequencing.</p>
<h2>Applications</h2>
<div align="center"><a href="https://www.diagenode.com/en/p/magmedip-kit-x48-48-rxns" class="center alert radius button"> qPCR analysis</a></div>
<div align="center"><a href="https://www.diagenode.com/en/p/magmedip-seq-package-V2-x10" class="center alert radius button"> NGS analysis </a></div>
<h2>Advantages</h2>
<ul style="font-size: 19px;" class="nobullet">
<li><i class="fa fa-arrow-circle-right"></i> <strong>Unaffected</strong> DNA</li>
<li><i class="fa fa-arrow-circle-right"></i> <strong>High enrichment</strong> yield</li>
<li><i class="fa fa-arrow-circle-right"></i> <strong>Robust</strong> & <strong>reproducible</strong> techniques</li>
<li><i class="fa fa-arrow-circle-right"></i> <strong>NGS</strong> compatible</li>
</ul>
<h2></h2>
</div>
</div>
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<p>Sodium bisulfite conversion of genomic DNA is the most commonly used method for DNA methylation studies providing <strong>single nucleotide resolution</strong>. It enables <span>to differentiate and detect unmethylated versus methylated cytosines. This procedure can then be followed either by <strong>PCR amplification</strong> or <strong>next generation sequencing</strong> to reveal the methylation status of every cytosine in gene specific amplification or whole genome amplification.</span></p>
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<h2>How it works</h2>
<p style="text-align: left;">Treatment of DNA with sodium bisulfite converts unmethylated cytosine to uracil, while methylated cytosines remain unchanged. <span>The DNA is then amplified by PCR where the uracils are converted to thymines. </span></p>
<p style="text-align: center;"><span></span></p>
<p><img src="https://www.diagenode.com/img/categories/bisulfite-conversion/bisulfite-conversion-acgautac.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<h2>Advantages</h2>
<ul class="nobullet" style="font-size: 19px;">
<li><i class="fa fa-arrow-circle-right"></i><strong> </strong><strong>Single nucleotide</strong> resolution</li>
<li><i class="fa fa-arrow-circle-right"></i><strong> Gene-specific </strong>and <strong>genome-wide</strong><span> analyses</span></li>
<li><i class="fa fa-arrow-circle-right"></i><strong> NGS</strong><span> </span>compatible</li>
</ul>
<h2>Downstream analysis techniques</h2>
<ul class="square">
<li>Reduced Representation Bisulfite Sequencing (RRBS) with our <a href="https://www.diagenode.com/en/p/premium-rrbs-kit-V2-x24">Premium RRBS Kit V2</a></li>
<li>Bisulfite conversion with our <a href="https://www.diagenode.com/en/p/premium-bisulfite-kit-50-rxns">Premium Bisulfite Kit</a> followed by qPCR, Sanger, Pyrosequencing</li>
</ul>
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<p>The Methylated DNA Immunoprecipitation is based on the affinity purification of methylated and hydroxymethylated DNA using, respectively, an antibody directed against 5-methylcytosine (5-mC) in the case of MeDIP or 5-hydroxymethylcytosine (5-hmC) in the case of hMeDIP.</p>
<h2>How it works</h2>
<p>In brief, Methyl DNA IP is performed as follows: Genomic DNA from cultured cells or tissues is prepared, sheared, and then denatured. Then, immunoselection and immunoprecipitation can take place using the antibody directed against 5 methylcytosine and antibody binding beads. After isolation and purification is performed, the IP’d methylated DNA is ready for any subsequent analysis as qPCR, amplification, hybridization on microarrays or next generation sequencing.</p>
<h2>Applications</h2>
<div align="center"><a href="https://www.diagenode.com/en/p/magmedip-kit-x48-48-rxns" class="center alert radius button"> qPCR analysis</a></div>
<div align="center"><a href="https://www.diagenode.com/en/p/magmedip-kit-x48-48-rxns" class="center alert radius button"> NGS analysis </a></div>
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<li><i class="fa fa-arrow-circle-right"></i> <strong>High enrichment</strong> yield</li>
<li><i class="fa fa-arrow-circle-right"></i> <strong>Robust</strong> & <strong>reproducible</strong> techniques</li>
<li><i class="fa fa-arrow-circle-right"></i> <strong>NGS</strong> compatible</li>
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<li>RRBS_methylated_control.fa: the sequence of the methylated spike-in control in FASTA format</li>
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<p>The DNA methylation control package includes one methylated and one unmethylated spike-in controls together with their corresponding qPCR primer sets for assessing the efficiency of your non-plant MeDIP experiments (Methylated DNA Immunoprecipitation) carried out with Diagenode’s <a href="https://www.diagenode.com/en/p/magmedip-kit-x48-48-rxns">MagMeDIP qPCR Kit</a> and <a href="https://www.diagenode.com/en/p/auto-magmedip-kit-x48-48-rxns" target="_blank">Auto MagMeDIP qPCR Kit</a>.</p>
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<p><em><strong>CAUTION</strong>: These spike-in controls are compatible with <span>Diagenode’s </span><a href="https://www.diagenode.com/en/p/magmedip-kit-x48-48-rxns">MagMeDIP qPCR Kit</a><span><span> </span>and<span> </span></span><a href="https://www.diagenode.com/en/p/auto-magmedip-kit-x48-48-rxns" target="_blank">Auto MagMeDIP qPCR Kit</a><span>. However</span>, those are not the ones directly provided in the kits. The spike-in controls provided in the kits are available separately with the <a href="https://www.diagenode.com/en/p/dna-methylation-control-package-V2-48-rxns">DNA methylation control package V2</a>.</em></p>',
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<li>RRBS_methylated_control.fa: the sequence of the methylated spike-in control in FASTA format</li>
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<h1 class="advertising-feature"><strong>Premium RRBS technology: cost-effective DNA methylation mapping with superior coverage</strong></h1>
<ul class="citation dates">
<li style="text-align: left;" class="vcard last-author"><a href="#auth-5" class="name"><span class="fn">Christoph Bock</span></a><sup><a href="#a2">2</a></sup><sup href="#affil-auth">, </sup></li>
<li style="text-align: left;" class="vcard"><a href="#auth-4" class="name"><span class="fn">Sharon Squazzo</span></a><sup><a href="#a3">3</a></sup><sup>, </sup></li>
<li style="text-align: left;" class="vcard"><a href="#auth-3" class="name"><span class="fn">Miklos Laczik</span></a><sup><a href="#a1">1</a></sup><sup>, </sup></li>
<li style="text-align: left;" class="vcard"><a href="#auth-2" class="name"><span class="fn">Paul Datlinger</span></a><sup><a href="#a2">2</a></sup><sup>, </sup></li>
<li style="text-align: left;" class="vcard c1"><a href="#auth-1" class="name"><span class="fn">Anne-Clémence Veillard</span></a><sup><a href="#a1">1</a></sup><sup>, </sup></li>
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<p>Nature Methods 13 (2016)</p>
<p>Published online <time datetime="2016-01-28">28 January 2016 </time></p>
<p></p>
<p><a href="http://www.nature.com/nmeth/journal/v13/n2/full/nmeth.f.391.html" class="alert button"><span style="font-weight: 400;">CHECK OUT THE NATURE METHODS PAPER</span></a></p>',
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<li><strong>High throughput</strong> - 96 samples processed in one experiment</li>
<li><strong>Cost-efficient</strong> – Multiplex up to 6 samples/sequencing lane</li>
<li><strong>Validated</strong> with FFPE, cancer, and low-input samples</li>
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<div class="small-6 columns"><br />
<p><b>Superior coverage</b></p>
<p>Comparison of CpG coverage between competing technologies.</p>
<p><strong><em><small>They love it! </small></em></strong><br /><em><small>The new Diagenode Premium RRBS Kit makes it easy to use RRBS cost-effectively and with high throughput, using early sample pooling and multiplex sequencing. Most importantly, the method provides an improved coverage of up to 4 million CpGs for the human genome. We successfully used this protocol on more than 1,000 samples comprising of six different species, various cancers, FFPE and lowinput samples. <br /><strong>Paul Datlinger and Christoph Bock, </strong><strong>CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria </strong></small></em></p>
<p><em> </em></p>
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<div class="row">
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<div class="small-6 columns">
<p><b>Accurate determination of DNA methylation level</b></p>
Excellent results were obtained using Diagenode’s Premium RRBS kit: almost 90% alignment rate, 4.1 million CpGs covered and bisulfite conversion rates around 99.5% for all samples. DNA methylation percentages in the region of IGF2 obtained with the Diagenode’s Premium RRBS Kit. Two human cell lines were analyzed: Gm12878 and MCF7. The MCF7 cell line was studied in duplicates. Each peak represents the DNA methylation percentage at one CpG. The methylated CpGs are shown in red and the unmethylated CpGs in grey.
<p><br /><br /></p>
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<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/landing-pages/rrbs_how_it_works.jpg" alt="rrbs how it works" style="display: block; margin-left: auto; margin-right: auto;" /><br /><br /></p>
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<div class="small-6 columns">
<p><b>How it works</b></p>
By cutting the genome using the <strong>restriction enzyme MspI</strong> (CCGG target sites) followed by size selection, DNA is enriched to represent <strong>CpG-rich genomic regions</strong> (including CpG islands, CpG island shores, enhancers, and other gene-regulatory elements), which are particularly relevant for epigenetic regulation. Similar to exome-sequencing for mutation discovery, the RRBS protocol enriches for some of the most interesting target regions and thereby achieves a reduction in sequencing cost of a factor of 10-20 compared to whole genome bisulfite sequencing.</div>
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<table width="1039">
<tbody>
<tr></tr>
<tr>
<td width="281"> </td>
<td width="379">
<h4>Premium RRBS Kit</h4>
</td>
<td width="379">
<h5>Illumina® RRBS protocol</h5>
</td>
</tr>
<tr>
<td><strong>DNA input</strong></td>
<td>100 ng</td>
<td>2-5 µg</td>
</tr>
<tr>
<td><strong>Multiplexing</strong></td>
<td>Pooling of 6 samples (one HiSeq lane)</td>
<td>no guidelines</td>
</tr>
<tr>
<td><strong>Bisulfite conversion</strong></td>
<td>One bisulfite reaction per pool (6 samples)</td>
<td>One bisulfite reaction per sample</td>
</tr>
<tr>
<td><strong># purification steps</strong></td>
<td>2</td>
<td>7</td>
</tr>
<tr>
<td><strong>Key features</strong></td>
<td width="379">low input - cost-effective - optimized for high-throughput - complete kit</td>
<td width="379">high input - not optimized for high-throughput - reagents from different providers</td>
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<p></p>
<h1 class="advertising-feature"><strong>Premium RRBS technology: cost-effective DNA methylation mapping with superior coverage</strong></h1>
<ul class="citation dates">
<li style="text-align: left;" class="vcard last-author"><a href="#auth-5" class="name"><span class="fn">Christoph Bock</span></a><sup><a href="#a2">2</a></sup><sup href="#affil-auth">, </sup></li>
<li style="text-align: left;" class="vcard"><a href="#auth-4" class="name"><span class="fn">Sharon Squazzo</span></a><sup><a href="#a3">3</a></sup><sup>, </sup></li>
<li style="text-align: left;" class="vcard"><a href="#auth-3" class="name"><span class="fn">Miklos Laczik</span></a><sup><a href="#a1">1</a></sup><sup>, </sup></li>
<li style="text-align: left;" class="vcard"><a href="#auth-2" class="name"><span class="fn">Paul Datlinger</span></a><sup><a href="#a2">2</a></sup><sup>, </sup></li>
<li style="text-align: left;" class="vcard c1"><a href="#auth-1" class="name"><span class="fn">Anne-Clémence Veillard</span></a><sup><a href="#a1">1</a></sup><sup>, </sup></li>
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<p>Nature Methods 13 (2016)</p>
<p>Published online <time datetime="2016-01-28">28 January 2016 </time></p>
<p></p>
<p><a href="http://www.nature.com/nmeth/journal/v13/n2/full/nmeth.f.391.html" class="alert button"><span style="font-weight: 400;">CHECK OUT THE NATURE METHODS PAPER</span></a></p>
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<p> </p>
<div class="small-12 medium-4 large-4 columns"><center></center><center></center><center></center><center><a href="https://www.ncbi.nlm.nih.gov/pubmed/30429608" target="_blank"><img src="https://www.diagenode.com/img/banners/banner-nature-publication-580.png" alt="Click here to read more about MeDIP " caption="false" width="80%" /></a></center></div>
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<h3 style="text-align: justify;">Sensitive tumour detection and classification using plasma cell-free DNA methylomes<br /><a href="https://www.ncbi.nlm.nih.gov/pubmed/30429608" target="_blank">Read the publication</a></h3>
<h3 class="c-article-title u-h1" data-test="article-title" itemprop="name headline" style="text-align: justify;">Preparation of cfMeDIP-seq libraries for methylome profiling of plasma cell-free DNA<br /><a href="https://www.nature.com/articles/s41596-019-0202-2" target="_blank" title="cfMeDIP-seq Nature Method">Read the method</a></h3>
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<p>Perform <strong>MeDIP</strong> (<strong>Me</strong>thylated <strong>D</strong>NA <strong>I</strong>mmuno<strong>p</strong>recipitation) followed by qPCR or NGS to estimate DNA methylation status of your sample using a highly sensitive 5-methylcytosine antibody. Our MagMeDIP kit contains high quality reagents to get the highest enrichment of methylated DNA with an optimized user-friendly protocol.</p>
</div>
</div>
<h3><span>Features</span></h3>
<ul>
<li>Starting DNA amount: <strong>10 ng – 1 µg</strong></li>
<li>Content: <strong>all reagents included</strong> for DNA extraction, immunoprecipitation (including the 5-mC antibody, spike-in controls and their corresponding qPCR primer pairs) as well as DNA isolation after IP.</li>
<li>Application: <strong>qPCR</strong> and <strong>NGS</strong></li>
<li>Robust method, <strong>superior enrichment</strong>, and easy-to-use protocol</li>
<li><strong>High reproducibility</strong> between replicates and repetitive experiments</li>
<li>Compatible with <strong>all species </strong></li>
</ul>',
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<h3><span>How it works</span></h3>
<p>In brief, after the cell collection and lysis, the genomic DNA is extracted, sheared, and then denatured. In the next step the antibody directed against 5 methylcytosine and antibody binding beads are used for immunoselection and immunoprecipitation of methylated DNA fragments. Then, the IP’d methylated DNA is isolated and can be used for any subsequent analysis as qPCR, amplification, hybridization on microarrays or next generation sequencing.</p>
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'info2' => '<p>The kit MagMeDIP contains all reagents necessary for a complete MeDIP-qPCR workflow. Two MagMeDIP protocols have been validated: for manual processing as well as for automated processing, using the Diagenode’s IP-Star Compact Automated System (please refer to the kit manual).</p>
<ul>
<li><strong>Complete kit</strong> including DNA extraction module, IP antibody and reagents, DNA isolation buffer</li>
<li><strong>Quality control of the IP:</strong> due to methylated and unmethylated DNA spike-in controls and their associated qPCR primers</li>
<li><strong>Easy to use</strong> with user-friendly magnetic beads and rack</li>
<li><strong>Highly validated protocol</strong></li>
<li>Automated protocol supplied</li>
</ul>
<center><img src="https://www.diagenode.com/img/product/kits/fig1-magmedipkit.png" width="85%" alt="Methylated DNA Immunoprecipitation" caption="false" /></center>
<p style="font-size: 0.9em;"><em><strong>Figure 1.</strong> Immunoprecipitation results obtained with Diagenode MagMeDIP Kit</em></p>
<p style="font-size: 0.9em;">MeDIP assays were performed manually using 1 µg or 50 ng gDNA from blood cells with the MagMeDIP kit (Diagenode). The IP was performed with the Methylated and Unmethylated spike-in controls included in the kit, together with the human DNA samples. The DNA was isolated/purified using DIB. Afterwards, qPCR was performed using the primer pairs included in this kit.</p>
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'info3' => '<p>For DNA methylation analysis on the whole genome, MagMeDIP kit can be coupled with Next-Generation Sequencing. To perform MeDIP-sequencing we recommend the following strategy:</p>
<ul style="list-style-type: circle;">
<li>Choose a library preparation solution which is compatible with the starting amount of DNA you are planning to use (from 10 ng to 1 μg). It can be a home-made solution or a commercial one.</li>
<li>Choose the indexing system that fits your needs considering the following features:</li>
<ul>
<ul>
<ul>
<li>Single-indexing, combinatorial dual-indexing or unique dual-indexing</li>
<li>Number of barcodes</li>
<li>Full-length adaptors containing the barcodes or barcoding at the final amplification step</li>
<li>Presence / absence of Unique Molecular Identifiers (for PCR duplicates removal)</li>
</ul>
</ul>
</ul>
<li>Standard library preparation protocols are compatible with double-stranded DNA only, therefore the first steps of the library preparation (end repair, A-tailing, adaptor ligation and clean-up) will have to be performed on sheared DNA, before the IP.</li>
</ul>
<p style="padding-left: 30px;"><strong>CAUTION:</strong> As the immunoprecipitation step occurs at the middle of the library preparation workflow, single-tube solutions for library preparation are usually not compatible with MeDIP-sequencing.</p>
<ul style="list-style-type: circle;">
<li>For DNA isolation after the IP, we recommend using the <a href="https://www.diagenode.com/en/p/ipure-kit-v2-x24" title="IPure kit v2">IPure kit v2</a> (available separately, Cat. No. C03010014) instead of DNA isolation Buffer.</li>
</ul>
<ul style="list-style-type: circle;">
<li>Perform library amplification after the DNA isolation following the standard protocol of the chosen library preparation solution.</li>
</ul>
<h3><span>MeDIP-seq workflow</span></h3>
<center><img src="https://www.diagenode.com/img/product/kits/MeDIP-seq-workflow.png" width="110%" alt="MagMeDIP qPCR Kit x10 workflow" caption="false" /></center>
<h3><span>Example of results</span></h3>
<center><img src="https://www.diagenode.com/img/product/kits/medip-specificity.png" alt="MagMeDIP qPCR Kit Result" caption="false" width="951" height="488" /></center>
<p></p>
<p style="font-size: 0.9em;"><strong>Figure 1. qPCR analysis of external spike-in DNA controls (methylated and unmethylated) after IP.</strong> Samples were prepared using 1μg – 100ng -10ng sheared human gDNA with the MagMeDIP kit (Diagenode) and a commercially available library prep kit. DNA isolation after IP has been performed with IPure kit V2 (Diagenode).</p>
<p></p>
<p></p>
<center><img src="https://www.diagenode.com/img/product/kits/medip-saturation-analysis.png" alt=" MagMeDIP kit " caption="false" width="951" height="461" /></center>
<p></p>
<p style="font-size: 0.9em;"><strong>Figure 2. Saturation analysis.</strong> Clean reads were aligned to the human genome (hg19) using Burrows-Wheeler aligner (BWA) algorithm after which duplicated and unmapped reads were removed resulting in a mapping efficiency >98% for all samples. Quality and validity check of the mapped MeDIP-seq data was performed using MEDIPS R package. Saturation plots show that all sets of reads have sufficient complexity and depth to saturate the coverage profile of the reference genome and that this is reproducible between replicates and repetitive experiments (data shown for 50 ng gDNA input: left panel = replicate a, right panel = replicate b).</p>
<p></p>
<p></p>
<center><img src="https://www.diagenode.com/img/product/kits/medip-libraries-prep.png" alt="MagMeDIP x10 " caption="false" width="951" height="708" /></center>
<p></p>
<p style="font-size: 0.9em;"><strong>Figure 3. Sequencing profiles of MeDIP-seq libraries prepared from different starting amounts of sheared gDNA on the positive and negative methylated control regions.</strong> MeDIP-seq libraries were prepared from decreasing starting amounts of gDNA (1 μg (green), 50 ng (red), and 10ng (blue)) originating from human blood with the MagMeDIP kit (Diagenode) and a commercially available library prep kit. DNA isolation after IP has been performed with IPure kit V2 (Diagenode). IP and corresponding INPUT samples were sequenced on Illumina NovaSeq SP with 2x50 PE reads. The reads were mapped to the human genome (hg19) with bwa and the alignments were loaded into IGV (the tracks use an identical scale). The top IGV figure shows the TSH2B (also known as H2BC1) gene (marked by blue boxes in the bottom track) and its surroundings. The TSH2B gene is coding for a histone variant that does not occur in blood cells, and it is known to be silenced by methylation. Accordingly, we see a high coverage in the vicinity of this gene. The bottom IGV figure shows the GADPH locus (marked by blue boxes in the bottom track) and its surroundings. The GADPH gene is a highly active transcription region and should not be methylated, resulting in no reads accumulation following MeDIP-seq experiment.</p>
<p></p>
<ul>
<ul>
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'meta_description' => 'Perform Methylated DNA Immunoprecipitation (MeDIP) to estimate DNA methylation status of your sample using highly specific 5-mC antibody. This kit allows the preparation of cfMeDIP-seq libraries.',
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'description' => '<p><a href="https://www.diagenode.com/files/products/kits/premium-RRBS-spike-in%20controls-08_18.pdf"><img src="https://www.diagenode.com/img/buttons/bt-manual.png" /></a></p>
<p>The DNA methylation control package includes one methylated and one unmethylated spike-in controls together with their corresponding qPCR primer sets for assessing the efficiency of your non-plant MeDIP experiments (Methylated DNA Immunoprecipitation) carried out with Diagenode’s <a href="https://www.diagenode.com/en/p/magmedip-kit-x48-48-rxns">MagMeDIP qPCR Kit</a> and <a href="https://www.diagenode.com/en/p/auto-magmedip-kit-x48-48-rxns" target="_blank">Auto MagMeDIP qPCR Kit</a>.</p>
<p><span>Those spike-in controls are made from <em>A. thaliana</em><span>. Therefore, they can interfere with DNA samples derived from plant species.</span></span></p>
<p><em><strong>CAUTION</strong>: These spike-in controls are compatible with <span>Diagenode’s </span><a href="https://www.diagenode.com/en/p/magmedip-kit-x48-48-rxns">MagMeDIP qPCR Kit</a><span><span> </span>and<span> </span></span><a href="https://www.diagenode.com/en/p/auto-magmedip-kit-x48-48-rxns" target="_blank">Auto MagMeDIP qPCR Kit</a><span>. However</span>, those are not the ones directly provided in the kits. The spike-in controls provided in the kits are available separately with the <a href="https://www.diagenode.com/en/p/dna-methylation-control-package-V2-48-rxns">DNA methylation control package V2</a>.</em></p>',
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'meta_description' => 'DNA Methylation control package',
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'description' => '<div class="row">
<div class="large-12 columns">
<div style="text-align: justify;" class="small-12 medium-8 large-8 columns">
<h2>Complete solutions for DNA methylation studies</h2>
<p>Whether you are experienced or new to the field of DNA methylation, Diagenode has everything you need to make your assay as easy and convenient as possible while ensuring consistent data between samples and experiments. Diagenode offers sonication instruments, reagent kits, high quality antibodies, and high-throughput automation capability to address all of your specific DNA methylation analysis requirements.</p>
</div>
<div class="small-12 medium-4 large-4 columns text-center"><a href="../landing-pages/dna-methylation-grant-applications"><img src="https://www.diagenode.com/img/banners/banner-dna-grant.png" alt="" /></a></div>
<div style="text-align: justify;" class="small-12 medium-12 large-12 columns">
<p>DNA methylation was the first discovered epigenetic mark and is the most widely studied topic in epigenetics. <em>In vivo</em>, DNA is methylated following DNA replication and is involved in a number of biological processes including the regulation of imprinted genes, X chromosome inactivation. and tumor suppressor gene silencing in cancer cells. Methylation often occurs in cytosine-guanine rich regions of DNA (CpG islands), which are commonly upstream of promoter regions.</p>
</div>
<div class="small-12 medium-12 large-12 columns"><br /><br />
<ul class="accordion" data-accordion="">
<li class="accordion-navigation"><a href="#dnamethyl"><i class="fa fa-caret-right"></i> Learn more</a>
<div id="dnamethyl" class="content">5-methylcytosine (5-mC) has been known for a long time as the only modification of DNA for epigenetic regulation. In 2009, however, Kriaucionis discovered a second methylated cytosine, 5-hydroxymethylcytosine (5-hmC). The so-called 6th base, is generated by enzymatic conversion of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine by the TET family of oxygenases. Early reports suggested that 5-hmC may represent an intermediate of active demethylation in a new pathway which demethylates DNA, converting 5-mC to cytosine. Recent evidence fuel this hypothesis suggesting that further oxidation of the hydroxymethyl group leads to a formyl or carboxyl group followed by either deformylation or decarboxylation. The formyl and carboxyl groups of 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) could be enzymatically removed without excision of the base.
<p class="text-center"><img src="https://www.diagenode.com/img/categories/kits_dna/dna_methylation_variants.jpg" /></p>
</div>
</li>
</ul>
<br />
<h2>Main DNA methylation technologies</h2>
<p style="text-align: justify;">Overview of the <span style="font-weight: 400;">three main approaches for studying DNA methylation.</span></p>
<div class="row">
<ol>
<li style="font-weight: 400;"><span style="font-weight: 400;">Chemical modification with bisulfite – Bisulfite conversion</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Enrichment of methylated DNA (including MeDIP and MBD)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Treatment with methylation-sensitive or dependent restriction enzymes</span></li>
</ol>
<p><span style="font-weight: 400;"> </span></p>
<div class="row">
<table>
<thead>
<tr>
<th></th>
<th>Description</th>
<th width="350">Features</th>
</tr>
</thead>
<tbody>
<tr>
<td><strong>Bisulfite conversion</strong></td>
<td><span style="font-weight: 400;">Chemical conversion of unmethylated cytosine to uracil. Methylated cytosines are protected from this conversion allowing to determine DNA methylation at single nucleotide resolution.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Single nucleotide resolution</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Quantitative analysis - methylation rate (%)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Gold standard and well studied</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Compatible with automation</span></li>
</ul>
</td>
</tr>
<tr>
<td><b>Methylated DNA enrichment</b></td>
<td><span style="font-weight: 400;">(Hydroxy-)Methylated DNA is enriched by using specific antibodies (hMeDIP or MeDIP) or proteins (MBD) that specifically bind methylated CpG sites in fragmented genomic DNA.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Resolution depends on the fragment size of the enriched methylated DNA (300 bp)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Qualitative analysis</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Compatible with automation</span></li>
</ul>
</td>
</tr>
<tr>
<td><strong>Restriction enzyme-based digestion</strong></td>
<td><span style="font-weight: 400;">Use of (hydroxy)methylation-sensitive or (hydroxy)methylation-dependent restriction enzymes for DNA methylation analysis at specific sites.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Determination of methylation status is limited by the enzyme recognition site</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Easy to use</span></li>
</ul>
</td>
</tr>
</tbody>
</table>
</div>
</div>
<div class="row"></div>
</div>
</div>
<div class="large-12 columns"></div>
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'meta_description' => 'Complete, optimized solutions for analyzing DNA methylation manually or on our automated system.',
'meta_title' => 'DNA Methylation - Bisulfite sequencing - Epigenetics | Diagenode',
'modified' => '2019-03-25 10:07:27',
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<div style="text-align: justify;" class="large-12 columns">Bisulfite modification of DNA is the most commonly used, "<strong>gold standard</strong>" method for DNA methylation studies providing <strong>single nucleotide resolution</strong>. T<span style="font-weight: 400;">his technology is based on the chemical conversion of unmethylated cytosine to uracil. Methylated cytosines are protected from this conversion allowing to determine DNA methylation at the singe nucleotide level.</span></div>
<div style="text-align: justify;" class="large-12 columns"></div>
<div style="text-align: justify;" class="large-12 columns">Various analyses can be performed on the altered sequence to retrieve this information: bisulfite sequencing, pyrosequencing, methylation-specific PCR, high resolution melting curve analysis, microarray-based approaches, and next-generation sequencing.
<h3>How it works</h3>
Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected (see Figure 1).
<p class="text-center"><img src="https://www.diagenode.com/img/applications/bisulfite.png" /><br />Figure 1: Overview of bisulfite conversion of DNA</p>
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'meta_title' => 'Bisulfite sequencing(Bis-Seq) - Bisulfite conversion - DNA Methylation | Diagenode',
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<div class="small-12 medium-3 large-3 columns"><center><a href="https://www.ncbi.nlm.nih.gov/pubmed/30429608" target="_blank"><img src="https://www.diagenode.com/img/banners/banner-nature-publication-580.png" /></a></center></div>
<div class="small-12 medium-9 large-9 columns">
<h3>Sensitive tumour detection and classification using plasma cell-free DNA methylomes<br /><a href="https://www.ncbi.nlm.nih.gov/pubmed/30429608" target="_blank">Read the publication</a></h3>
<h3 class="c-article-title u-h1" data-test="article-title" itemprop="name headline">Preparation of cfMeDIP-seq libraries for methylome profiling of plasma cell-free DNA<br /><a href="https://www.nature.com/articles/s41596-019-0202-2" target="_blank" title="cfMeDIP-seq Nature Method">Read the method</a></h3>
</div>
</div>
<div class="row">
<div class="large-12 columns"><span>The Methylated DNA Immunoprecipitation is based on the affinity purification of methylated and hydroxymethylated DNA using, respectively, an antibody directed against 5-methylcytosine (5-mC) in the case of MeDIP or 5-hydroxymethylcytosine (5-hmC) in the case of hMeDIP.</span><br />
<h2></h2>
<h2>How it works</h2>
<p>In brief, Methyl DNA IP is performed as follows: Genomic DNA from cultured cells or tissues is prepared, sheared, and then denatured. Then, immunoselection and immunoprecipitation can take place using the antibody directed against 5 methylcytosine and antibody binding beads. After isolation and purification is performed, the IP’d methylated DNA is ready for any subsequent analysis as qPCR, amplification, hybridization on microarrays or next generation sequencing.</p>
<h2>Applications</h2>
<div align="center"><a href="https://www.diagenode.com/en/p/magmedip-kit-x48-48-rxns" class="center alert radius button"> qPCR analysis</a></div>
<div align="center"><a href="https://www.diagenode.com/en/p/magmedip-seq-package-V2-x10" class="center alert radius button"> NGS analysis </a></div>
<h2>Advantages</h2>
<ul style="font-size: 19px;" class="nobullet">
<li><i class="fa fa-arrow-circle-right"></i> <strong>Unaffected</strong> DNA</li>
<li><i class="fa fa-arrow-circle-right"></i> <strong>High enrichment</strong> yield</li>
<li><i class="fa fa-arrow-circle-right"></i> <strong>Robust</strong> & <strong>reproducible</strong> techniques</li>
<li><i class="fa fa-arrow-circle-right"></i> <strong>NGS</strong> compatible</li>
</ul>
<h2></h2>
</div>
</div>
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<p>Sodium bisulfite conversion of genomic DNA is the most commonly used method for DNA methylation studies providing <strong>single nucleotide resolution</strong>. It enables <span>to differentiate and detect unmethylated versus methylated cytosines. This procedure can then be followed either by <strong>PCR amplification</strong> or <strong>next generation sequencing</strong> to reveal the methylation status of every cytosine in gene specific amplification or whole genome amplification.</span></p>
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<h2>How it works</h2>
<p style="text-align: left;">Treatment of DNA with sodium bisulfite converts unmethylated cytosine to uracil, while methylated cytosines remain unchanged. <span>The DNA is then amplified by PCR where the uracils are converted to thymines. </span></p>
<p style="text-align: center;"><span></span></p>
<p><img src="https://www.diagenode.com/img/categories/bisulfite-conversion/bisulfite-conversion-acgautac.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<h2>Advantages</h2>
<ul class="nobullet" style="font-size: 19px;">
<li><i class="fa fa-arrow-circle-right"></i><strong> </strong><strong>Single nucleotide</strong> resolution</li>
<li><i class="fa fa-arrow-circle-right"></i><strong> Gene-specific </strong>and <strong>genome-wide</strong><span> analyses</span></li>
<li><i class="fa fa-arrow-circle-right"></i><strong> NGS</strong><span> </span>compatible</li>
</ul>
<h2>Downstream analysis techniques</h2>
<ul class="square">
<li>Reduced Representation Bisulfite Sequencing (RRBS) with our <a href="https://www.diagenode.com/en/p/premium-rrbs-kit-V2-x24">Premium RRBS Kit V2</a></li>
<li>Bisulfite conversion with our <a href="https://www.diagenode.com/en/p/premium-bisulfite-kit-50-rxns">Premium Bisulfite Kit</a> followed by qPCR, Sanger, Pyrosequencing</li>
</ul>
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<p>The Methylated DNA Immunoprecipitation is based on the affinity purification of methylated and hydroxymethylated DNA using, respectively, an antibody directed against 5-methylcytosine (5-mC) in the case of MeDIP or 5-hydroxymethylcytosine (5-hmC) in the case of hMeDIP.</p>
<h2>How it works</h2>
<p>In brief, Methyl DNA IP is performed as follows: Genomic DNA from cultured cells or tissues is prepared, sheared, and then denatured. Then, immunoselection and immunoprecipitation can take place using the antibody directed against 5 methylcytosine and antibody binding beads. After isolation and purification is performed, the IP’d methylated DNA is ready for any subsequent analysis as qPCR, amplification, hybridization on microarrays or next generation sequencing.</p>
<h2>Applications</h2>
<div align="center"><a href="https://www.diagenode.com/en/p/magmedip-kit-x48-48-rxns" class="center alert radius button"> qPCR analysis</a></div>
<div align="center"><a href="https://www.diagenode.com/en/p/magmedip-kit-x48-48-rxns" class="center alert radius button"> NGS analysis </a></div>
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<li><i class="fa fa-arrow-circle-right"></i> <strong>High enrichment</strong> yield</li>
<li><i class="fa fa-arrow-circle-right"></i> <strong>Robust</strong> & <strong>reproducible</strong> techniques</li>
<li><i class="fa fa-arrow-circle-right"></i> <strong>NGS</strong> compatible</li>
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<li>RRBS_methylated_control.fa: the sequence of the methylated spike-in control in FASTA format</li>
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<p>The DNA methylation control package includes one methylated and one unmethylated spike-in controls together with their corresponding qPCR primer sets for assessing the efficiency of your non-plant MeDIP experiments (Methylated DNA Immunoprecipitation) carried out with Diagenode’s <a href="https://www.diagenode.com/en/p/magmedip-kit-x48-48-rxns">MagMeDIP qPCR Kit</a> and <a href="https://www.diagenode.com/en/p/auto-magmedip-kit-x48-48-rxns" target="_blank">Auto MagMeDIP qPCR Kit</a>.</p>
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<p><em><strong>CAUTION</strong>: These spike-in controls are compatible with <span>Diagenode’s </span><a href="https://www.diagenode.com/en/p/magmedip-kit-x48-48-rxns">MagMeDIP qPCR Kit</a><span><span> </span>and<span> </span></span><a href="https://www.diagenode.com/en/p/auto-magmedip-kit-x48-48-rxns" target="_blank">Auto MagMeDIP qPCR Kit</a><span>. However</span>, those are not the ones directly provided in the kits. The spike-in controls provided in the kits are available separately with the <a href="https://www.diagenode.com/en/p/dna-methylation-control-package-V2-48-rxns">DNA methylation control package V2</a>.</em></p>',
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<li>RRBS_methylated_control.fa: the sequence of the methylated spike-in control in FASTA format</li>
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<h1 class="advertising-feature"><strong>Premium RRBS technology: cost-effective DNA methylation mapping with superior coverage</strong></h1>
<ul class="citation dates">
<li style="text-align: left;" class="vcard last-author"><a href="#auth-5" class="name"><span class="fn">Christoph Bock</span></a><sup><a href="#a2">2</a></sup><sup href="#affil-auth">, </sup></li>
<li style="text-align: left;" class="vcard"><a href="#auth-4" class="name"><span class="fn">Sharon Squazzo</span></a><sup><a href="#a3">3</a></sup><sup>, </sup></li>
<li style="text-align: left;" class="vcard"><a href="#auth-3" class="name"><span class="fn">Miklos Laczik</span></a><sup><a href="#a1">1</a></sup><sup>, </sup></li>
<li style="text-align: left;" class="vcard"><a href="#auth-2" class="name"><span class="fn">Paul Datlinger</span></a><sup><a href="#a2">2</a></sup><sup>, </sup></li>
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<p>Published online <time datetime="2016-01-28">28 January 2016 </time></p>
<p></p>
<p><a href="http://www.nature.com/nmeth/journal/v13/n2/full/nmeth.f.391.html" class="alert button"><span style="font-weight: 400;">CHECK OUT THE NATURE METHODS PAPER</span></a></p>',
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<li><strong>High throughput</strong> - 96 samples processed in one experiment</li>
<li><strong>Cost-efficient</strong> – Multiplex up to 6 samples/sequencing lane</li>
<li><strong>Validated</strong> with FFPE, cancer, and low-input samples</li>
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<div class="small-6 columns"><br />
<p><b>Superior coverage</b></p>
<p>Comparison of CpG coverage between competing technologies.</p>
<p><strong><em><small>They love it! </small></em></strong><br /><em><small>The new Diagenode Premium RRBS Kit makes it easy to use RRBS cost-effectively and with high throughput, using early sample pooling and multiplex sequencing. Most importantly, the method provides an improved coverage of up to 4 million CpGs for the human genome. We successfully used this protocol on more than 1,000 samples comprising of six different species, various cancers, FFPE and lowinput samples. <br /><strong>Paul Datlinger and Christoph Bock, </strong><strong>CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria </strong></small></em></p>
<p><em> </em></p>
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<div class="small-6 columns">
<p><b>Accurate determination of DNA methylation level</b></p>
Excellent results were obtained using Diagenode’s Premium RRBS kit: almost 90% alignment rate, 4.1 million CpGs covered and bisulfite conversion rates around 99.5% for all samples. DNA methylation percentages in the region of IGF2 obtained with the Diagenode’s Premium RRBS Kit. Two human cell lines were analyzed: Gm12878 and MCF7. The MCF7 cell line was studied in duplicates. Each peak represents the DNA methylation percentage at one CpG. The methylated CpGs are shown in red and the unmethylated CpGs in grey.
<p><br /><br /></p>
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<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/landing-pages/rrbs_how_it_works.jpg" alt="rrbs how it works" style="display: block; margin-left: auto; margin-right: auto;" /><br /><br /></p>
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<div class="small-6 columns">
<p><b>How it works</b></p>
By cutting the genome using the <strong>restriction enzyme MspI</strong> (CCGG target sites) followed by size selection, DNA is enriched to represent <strong>CpG-rich genomic regions</strong> (including CpG islands, CpG island shores, enhancers, and other gene-regulatory elements), which are particularly relevant for epigenetic regulation. Similar to exome-sequencing for mutation discovery, the RRBS protocol enriches for some of the most interesting target regions and thereby achieves a reduction in sequencing cost of a factor of 10-20 compared to whole genome bisulfite sequencing.</div>
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<table width="1039">
<tbody>
<tr></tr>
<tr>
<td width="281"> </td>
<td width="379">
<h4>Premium RRBS Kit</h4>
</td>
<td width="379">
<h5>Illumina® RRBS protocol</h5>
</td>
</tr>
<tr>
<td><strong>DNA input</strong></td>
<td>100 ng</td>
<td>2-5 µg</td>
</tr>
<tr>
<td><strong>Multiplexing</strong></td>
<td>Pooling of 6 samples (one HiSeq lane)</td>
<td>no guidelines</td>
</tr>
<tr>
<td><strong>Bisulfite conversion</strong></td>
<td>One bisulfite reaction per pool (6 samples)</td>
<td>One bisulfite reaction per sample</td>
</tr>
<tr>
<td><strong># purification steps</strong></td>
<td>2</td>
<td>7</td>
</tr>
<tr>
<td><strong>Key features</strong></td>
<td width="379">low input - cost-effective - optimized for high-throughput - complete kit</td>
<td width="379">high input - not optimized for high-throughput - reagents from different providers</td>
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<p></p>
<h1 class="advertising-feature"><strong>Premium RRBS technology: cost-effective DNA methylation mapping with superior coverage</strong></h1>
<ul class="citation dates">
<li style="text-align: left;" class="vcard last-author"><a href="#auth-5" class="name"><span class="fn">Christoph Bock</span></a><sup><a href="#a2">2</a></sup><sup href="#affil-auth">, </sup></li>
<li style="text-align: left;" class="vcard"><a href="#auth-4" class="name"><span class="fn">Sharon Squazzo</span></a><sup><a href="#a3">3</a></sup><sup>, </sup></li>
<li style="text-align: left;" class="vcard"><a href="#auth-3" class="name"><span class="fn">Miklos Laczik</span></a><sup><a href="#a1">1</a></sup><sup>, </sup></li>
<li style="text-align: left;" class="vcard"><a href="#auth-2" class="name"><span class="fn">Paul Datlinger</span></a><sup><a href="#a2">2</a></sup><sup>, </sup></li>
<li style="text-align: left;" class="vcard c1"><a href="#auth-1" class="name"><span class="fn">Anne-Clémence Veillard</span></a><sup><a href="#a1">1</a></sup><sup>, </sup></li>
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<p>Nature Methods 13 (2016)</p>
<p>Published online <time datetime="2016-01-28">28 January 2016 </time></p>
<p></p>
<p><a href="http://www.nature.com/nmeth/journal/v13/n2/full/nmeth.f.391.html" class="alert button"><span style="font-weight: 400;">CHECK OUT THE NATURE METHODS PAPER</span></a></p>
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<p> </p>
<div class="small-12 medium-4 large-4 columns"><center></center><center></center><center></center><center><a href="https://www.ncbi.nlm.nih.gov/pubmed/30429608" target="_blank"><img src="https://www.diagenode.com/img/banners/banner-nature-publication-580.png" alt="Click here to read more about MeDIP " caption="false" width="80%" /></a></center></div>
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<h3 style="text-align: justify;">Sensitive tumour detection and classification using plasma cell-free DNA methylomes<br /><a href="https://www.ncbi.nlm.nih.gov/pubmed/30429608" target="_blank">Read the publication</a></h3>
<h3 class="c-article-title u-h1" data-test="article-title" itemprop="name headline" style="text-align: justify;">Preparation of cfMeDIP-seq libraries for methylome profiling of plasma cell-free DNA<br /><a href="https://www.nature.com/articles/s41596-019-0202-2" target="_blank" title="cfMeDIP-seq Nature Method">Read the method</a></h3>
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<p>Perform <strong>MeDIP</strong> (<strong>Me</strong>thylated <strong>D</strong>NA <strong>I</strong>mmuno<strong>p</strong>recipitation) followed by qPCR or NGS to estimate DNA methylation status of your sample using a highly sensitive 5-methylcytosine antibody. Our MagMeDIP kit contains high quality reagents to get the highest enrichment of methylated DNA with an optimized user-friendly protocol.</p>
</div>
</div>
<h3><span>Features</span></h3>
<ul>
<li>Starting DNA amount: <strong>10 ng – 1 µg</strong></li>
<li>Content: <strong>all reagents included</strong> for DNA extraction, immunoprecipitation (including the 5-mC antibody, spike-in controls and their corresponding qPCR primer pairs) as well as DNA isolation after IP.</li>
<li>Application: <strong>qPCR</strong> and <strong>NGS</strong></li>
<li>Robust method, <strong>superior enrichment</strong>, and easy-to-use protocol</li>
<li><strong>High reproducibility</strong> between replicates and repetitive experiments</li>
<li>Compatible with <strong>all species </strong></li>
</ul>',
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<h3><span>How it works</span></h3>
<p>In brief, after the cell collection and lysis, the genomic DNA is extracted, sheared, and then denatured. In the next step the antibody directed against 5 methylcytosine and antibody binding beads are used for immunoselection and immunoprecipitation of methylated DNA fragments. Then, the IP’d methylated DNA is isolated and can be used for any subsequent analysis as qPCR, amplification, hybridization on microarrays or next generation sequencing.</p>
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'info2' => '<p>The kit MagMeDIP contains all reagents necessary for a complete MeDIP-qPCR workflow. Two MagMeDIP protocols have been validated: for manual processing as well as for automated processing, using the Diagenode’s IP-Star Compact Automated System (please refer to the kit manual).</p>
<ul>
<li><strong>Complete kit</strong> including DNA extraction module, IP antibody and reagents, DNA isolation buffer</li>
<li><strong>Quality control of the IP:</strong> due to methylated and unmethylated DNA spike-in controls and their associated qPCR primers</li>
<li><strong>Easy to use</strong> with user-friendly magnetic beads and rack</li>
<li><strong>Highly validated protocol</strong></li>
<li>Automated protocol supplied</li>
</ul>
<center><img src="https://www.diagenode.com/img/product/kits/fig1-magmedipkit.png" width="85%" alt="Methylated DNA Immunoprecipitation" caption="false" /></center>
<p style="font-size: 0.9em;"><em><strong>Figure 1.</strong> Immunoprecipitation results obtained with Diagenode MagMeDIP Kit</em></p>
<p style="font-size: 0.9em;">MeDIP assays were performed manually using 1 µg or 50 ng gDNA from blood cells with the MagMeDIP kit (Diagenode). The IP was performed with the Methylated and Unmethylated spike-in controls included in the kit, together with the human DNA samples. The DNA was isolated/purified using DIB. Afterwards, qPCR was performed using the primer pairs included in this kit.</p>
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'info3' => '<p>For DNA methylation analysis on the whole genome, MagMeDIP kit can be coupled with Next-Generation Sequencing. To perform MeDIP-sequencing we recommend the following strategy:</p>
<ul style="list-style-type: circle;">
<li>Choose a library preparation solution which is compatible with the starting amount of DNA you are planning to use (from 10 ng to 1 μg). It can be a home-made solution or a commercial one.</li>
<li>Choose the indexing system that fits your needs considering the following features:</li>
<ul>
<ul>
<ul>
<li>Single-indexing, combinatorial dual-indexing or unique dual-indexing</li>
<li>Number of barcodes</li>
<li>Full-length adaptors containing the barcodes or barcoding at the final amplification step</li>
<li>Presence / absence of Unique Molecular Identifiers (for PCR duplicates removal)</li>
</ul>
</ul>
</ul>
<li>Standard library preparation protocols are compatible with double-stranded DNA only, therefore the first steps of the library preparation (end repair, A-tailing, adaptor ligation and clean-up) will have to be performed on sheared DNA, before the IP.</li>
</ul>
<p style="padding-left: 30px;"><strong>CAUTION:</strong> As the immunoprecipitation step occurs at the middle of the library preparation workflow, single-tube solutions for library preparation are usually not compatible with MeDIP-sequencing.</p>
<ul style="list-style-type: circle;">
<li>For DNA isolation after the IP, we recommend using the <a href="https://www.diagenode.com/en/p/ipure-kit-v2-x24" title="IPure kit v2">IPure kit v2</a> (available separately, Cat. No. C03010014) instead of DNA isolation Buffer.</li>
</ul>
<ul style="list-style-type: circle;">
<li>Perform library amplification after the DNA isolation following the standard protocol of the chosen library preparation solution.</li>
</ul>
<h3><span>MeDIP-seq workflow</span></h3>
<center><img src="https://www.diagenode.com/img/product/kits/MeDIP-seq-workflow.png" width="110%" alt="MagMeDIP qPCR Kit x10 workflow" caption="false" /></center>
<h3><span>Example of results</span></h3>
<center><img src="https://www.diagenode.com/img/product/kits/medip-specificity.png" alt="MagMeDIP qPCR Kit Result" caption="false" width="951" height="488" /></center>
<p></p>
<p style="font-size: 0.9em;"><strong>Figure 1. qPCR analysis of external spike-in DNA controls (methylated and unmethylated) after IP.</strong> Samples were prepared using 1μg – 100ng -10ng sheared human gDNA with the MagMeDIP kit (Diagenode) and a commercially available library prep kit. DNA isolation after IP has been performed with IPure kit V2 (Diagenode).</p>
<p></p>
<p></p>
<center><img src="https://www.diagenode.com/img/product/kits/medip-saturation-analysis.png" alt=" MagMeDIP kit " caption="false" width="951" height="461" /></center>
<p></p>
<p style="font-size: 0.9em;"><strong>Figure 2. Saturation analysis.</strong> Clean reads were aligned to the human genome (hg19) using Burrows-Wheeler aligner (BWA) algorithm after which duplicated and unmapped reads were removed resulting in a mapping efficiency >98% for all samples. Quality and validity check of the mapped MeDIP-seq data was performed using MEDIPS R package. Saturation plots show that all sets of reads have sufficient complexity and depth to saturate the coverage profile of the reference genome and that this is reproducible between replicates and repetitive experiments (data shown for 50 ng gDNA input: left panel = replicate a, right panel = replicate b).</p>
<p></p>
<p></p>
<center><img src="https://www.diagenode.com/img/product/kits/medip-libraries-prep.png" alt="MagMeDIP x10 " caption="false" width="951" height="708" /></center>
<p></p>
<p style="font-size: 0.9em;"><strong>Figure 3. Sequencing profiles of MeDIP-seq libraries prepared from different starting amounts of sheared gDNA on the positive and negative methylated control regions.</strong> MeDIP-seq libraries were prepared from decreasing starting amounts of gDNA (1 μg (green), 50 ng (red), and 10ng (blue)) originating from human blood with the MagMeDIP kit (Diagenode) and a commercially available library prep kit. DNA isolation after IP has been performed with IPure kit V2 (Diagenode). IP and corresponding INPUT samples were sequenced on Illumina NovaSeq SP with 2x50 PE reads. The reads were mapped to the human genome (hg19) with bwa and the alignments were loaded into IGV (the tracks use an identical scale). The top IGV figure shows the TSH2B (also known as H2BC1) gene (marked by blue boxes in the bottom track) and its surroundings. The TSH2B gene is coding for a histone variant that does not occur in blood cells, and it is known to be silenced by methylation. Accordingly, we see a high coverage in the vicinity of this gene. The bottom IGV figure shows the GADPH locus (marked by blue boxes in the bottom track) and its surroundings. The GADPH gene is a highly active transcription region and should not be methylated, resulting in no reads accumulation following MeDIP-seq experiment.</p>
<p></p>
<ul>
<ul>
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'meta_description' => 'Perform Methylated DNA Immunoprecipitation (MeDIP) to estimate DNA methylation status of your sample using highly specific 5-mC antibody. This kit allows the preparation of cfMeDIP-seq libraries.',
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'description' => '<p><a href="https://www.diagenode.com/files/products/kits/premium-RRBS-spike-in%20controls-08_18.pdf"><img src="https://www.diagenode.com/img/buttons/bt-manual.png" /></a></p>
<p>The DNA methylation control package includes one methylated and one unmethylated spike-in controls together with their corresponding qPCR primer sets for assessing the efficiency of your non-plant MeDIP experiments (Methylated DNA Immunoprecipitation) carried out with Diagenode’s <a href="https://www.diagenode.com/en/p/magmedip-kit-x48-48-rxns">MagMeDIP qPCR Kit</a> and <a href="https://www.diagenode.com/en/p/auto-magmedip-kit-x48-48-rxns" target="_blank">Auto MagMeDIP qPCR Kit</a>.</p>
<p><span>Those spike-in controls are made from <em>A. thaliana</em><span>. Therefore, they can interfere with DNA samples derived from plant species.</span></span></p>
<p><em><strong>CAUTION</strong>: These spike-in controls are compatible with <span>Diagenode’s </span><a href="https://www.diagenode.com/en/p/magmedip-kit-x48-48-rxns">MagMeDIP qPCR Kit</a><span><span> </span>and<span> </span></span><a href="https://www.diagenode.com/en/p/auto-magmedip-kit-x48-48-rxns" target="_blank">Auto MagMeDIP qPCR Kit</a><span>. However</span>, those are not the ones directly provided in the kits. The spike-in controls provided in the kits are available separately with the <a href="https://www.diagenode.com/en/p/dna-methylation-control-package-V2-48-rxns">DNA methylation control package V2</a>.</em></p>',
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'meta_description' => 'DNA Methylation control package',
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'description' => '<div class="row">
<div class="large-12 columns">
<div style="text-align: justify;" class="small-12 medium-8 large-8 columns">
<h2>Complete solutions for DNA methylation studies</h2>
<p>Whether you are experienced or new to the field of DNA methylation, Diagenode has everything you need to make your assay as easy and convenient as possible while ensuring consistent data between samples and experiments. Diagenode offers sonication instruments, reagent kits, high quality antibodies, and high-throughput automation capability to address all of your specific DNA methylation analysis requirements.</p>
</div>
<div class="small-12 medium-4 large-4 columns text-center"><a href="../landing-pages/dna-methylation-grant-applications"><img src="https://www.diagenode.com/img/banners/banner-dna-grant.png" alt="" /></a></div>
<div style="text-align: justify;" class="small-12 medium-12 large-12 columns">
<p>DNA methylation was the first discovered epigenetic mark and is the most widely studied topic in epigenetics. <em>In vivo</em>, DNA is methylated following DNA replication and is involved in a number of biological processes including the regulation of imprinted genes, X chromosome inactivation. and tumor suppressor gene silencing in cancer cells. Methylation often occurs in cytosine-guanine rich regions of DNA (CpG islands), which are commonly upstream of promoter regions.</p>
</div>
<div class="small-12 medium-12 large-12 columns"><br /><br />
<ul class="accordion" data-accordion="">
<li class="accordion-navigation"><a href="#dnamethyl"><i class="fa fa-caret-right"></i> Learn more</a>
<div id="dnamethyl" class="content">5-methylcytosine (5-mC) has been known for a long time as the only modification of DNA for epigenetic regulation. In 2009, however, Kriaucionis discovered a second methylated cytosine, 5-hydroxymethylcytosine (5-hmC). The so-called 6th base, is generated by enzymatic conversion of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine by the TET family of oxygenases. Early reports suggested that 5-hmC may represent an intermediate of active demethylation in a new pathway which demethylates DNA, converting 5-mC to cytosine. Recent evidence fuel this hypothesis suggesting that further oxidation of the hydroxymethyl group leads to a formyl or carboxyl group followed by either deformylation or decarboxylation. The formyl and carboxyl groups of 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) could be enzymatically removed without excision of the base.
<p class="text-center"><img src="https://www.diagenode.com/img/categories/kits_dna/dna_methylation_variants.jpg" /></p>
</div>
</li>
</ul>
<br />
<h2>Main DNA methylation technologies</h2>
<p style="text-align: justify;">Overview of the <span style="font-weight: 400;">three main approaches for studying DNA methylation.</span></p>
<div class="row">
<ol>
<li style="font-weight: 400;"><span style="font-weight: 400;">Chemical modification with bisulfite – Bisulfite conversion</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Enrichment of methylated DNA (including MeDIP and MBD)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Treatment with methylation-sensitive or dependent restriction enzymes</span></li>
</ol>
<p><span style="font-weight: 400;"> </span></p>
<div class="row">
<table>
<thead>
<tr>
<th></th>
<th>Description</th>
<th width="350">Features</th>
</tr>
</thead>
<tbody>
<tr>
<td><strong>Bisulfite conversion</strong></td>
<td><span style="font-weight: 400;">Chemical conversion of unmethylated cytosine to uracil. Methylated cytosines are protected from this conversion allowing to determine DNA methylation at single nucleotide resolution.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Single nucleotide resolution</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Quantitative analysis - methylation rate (%)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Gold standard and well studied</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Compatible with automation</span></li>
</ul>
</td>
</tr>
<tr>
<td><b>Methylated DNA enrichment</b></td>
<td><span style="font-weight: 400;">(Hydroxy-)Methylated DNA is enriched by using specific antibodies (hMeDIP or MeDIP) or proteins (MBD) that specifically bind methylated CpG sites in fragmented genomic DNA.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Resolution depends on the fragment size of the enriched methylated DNA (300 bp)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Qualitative analysis</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Compatible with automation</span></li>
</ul>
</td>
</tr>
<tr>
<td><strong>Restriction enzyme-based digestion</strong></td>
<td><span style="font-weight: 400;">Use of (hydroxy)methylation-sensitive or (hydroxy)methylation-dependent restriction enzymes for DNA methylation analysis at specific sites.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Determination of methylation status is limited by the enzyme recognition site</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Easy to use</span></li>
</ul>
</td>
</tr>
</tbody>
</table>
</div>
</div>
<div class="row"></div>
</div>
</div>
<div class="large-12 columns"></div>
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'meta_description' => 'Complete, optimized solutions for analyzing DNA methylation manually or on our automated system.',
'meta_title' => 'DNA Methylation - Bisulfite sequencing - Epigenetics | Diagenode',
'modified' => '2019-03-25 10:07:27',
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<div style="text-align: justify;" class="large-12 columns">Bisulfite modification of DNA is the most commonly used, "<strong>gold standard</strong>" method for DNA methylation studies providing <strong>single nucleotide resolution</strong>. T<span style="font-weight: 400;">his technology is based on the chemical conversion of unmethylated cytosine to uracil. Methylated cytosines are protected from this conversion allowing to determine DNA methylation at the singe nucleotide level.</span></div>
<div style="text-align: justify;" class="large-12 columns"></div>
<div style="text-align: justify;" class="large-12 columns">Various analyses can be performed on the altered sequence to retrieve this information: bisulfite sequencing, pyrosequencing, methylation-specific PCR, high resolution melting curve analysis, microarray-based approaches, and next-generation sequencing.
<h3>How it works</h3>
Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected (see Figure 1).
<p class="text-center"><img src="https://www.diagenode.com/img/applications/bisulfite.png" /><br />Figure 1: Overview of bisulfite conversion of DNA</p>
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'meta_title' => 'Bisulfite sequencing(Bis-Seq) - Bisulfite conversion - DNA Methylation | Diagenode',
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<div class="small-12 medium-3 large-3 columns"><center><a href="https://www.ncbi.nlm.nih.gov/pubmed/30429608" target="_blank"><img src="https://www.diagenode.com/img/banners/banner-nature-publication-580.png" /></a></center></div>
<div class="small-12 medium-9 large-9 columns">
<h3>Sensitive tumour detection and classification using plasma cell-free DNA methylomes<br /><a href="https://www.ncbi.nlm.nih.gov/pubmed/30429608" target="_blank">Read the publication</a></h3>
<h3 class="c-article-title u-h1" data-test="article-title" itemprop="name headline">Preparation of cfMeDIP-seq libraries for methylome profiling of plasma cell-free DNA<br /><a href="https://www.nature.com/articles/s41596-019-0202-2" target="_blank" title="cfMeDIP-seq Nature Method">Read the method</a></h3>
</div>
</div>
<div class="row">
<div class="large-12 columns"><span>The Methylated DNA Immunoprecipitation is based on the affinity purification of methylated and hydroxymethylated DNA using, respectively, an antibody directed against 5-methylcytosine (5-mC) in the case of MeDIP or 5-hydroxymethylcytosine (5-hmC) in the case of hMeDIP.</span><br />
<h2></h2>
<h2>How it works</h2>
<p>In brief, Methyl DNA IP is performed as follows: Genomic DNA from cultured cells or tissues is prepared, sheared, and then denatured. Then, immunoselection and immunoprecipitation can take place using the antibody directed against 5 methylcytosine and antibody binding beads. After isolation and purification is performed, the IP’d methylated DNA is ready for any subsequent analysis as qPCR, amplification, hybridization on microarrays or next generation sequencing.</p>
<h2>Applications</h2>
<div align="center"><a href="https://www.diagenode.com/en/p/magmedip-kit-x48-48-rxns" class="center alert radius button"> qPCR analysis</a></div>
<div align="center"><a href="https://www.diagenode.com/en/p/magmedip-seq-package-V2-x10" class="center alert radius button"> NGS analysis </a></div>
<h2>Advantages</h2>
<ul style="font-size: 19px;" class="nobullet">
<li><i class="fa fa-arrow-circle-right"></i> <strong>Unaffected</strong> DNA</li>
<li><i class="fa fa-arrow-circle-right"></i> <strong>High enrichment</strong> yield</li>
<li><i class="fa fa-arrow-circle-right"></i> <strong>Robust</strong> & <strong>reproducible</strong> techniques</li>
<li><i class="fa fa-arrow-circle-right"></i> <strong>NGS</strong> compatible</li>
</ul>
<h2></h2>
</div>
</div>
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<p>Sodium bisulfite conversion of genomic DNA is the most commonly used method for DNA methylation studies providing <strong>single nucleotide resolution</strong>. It enables <span>to differentiate and detect unmethylated versus methylated cytosines. This procedure can then be followed either by <strong>PCR amplification</strong> or <strong>next generation sequencing</strong> to reveal the methylation status of every cytosine in gene specific amplification or whole genome amplification.</span></p>
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<h2>How it works</h2>
<p style="text-align: left;">Treatment of DNA with sodium bisulfite converts unmethylated cytosine to uracil, while methylated cytosines remain unchanged. <span>The DNA is then amplified by PCR where the uracils are converted to thymines. </span></p>
<p style="text-align: center;"><span></span></p>
<p><img src="https://www.diagenode.com/img/categories/bisulfite-conversion/bisulfite-conversion-acgautac.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<h2>Advantages</h2>
<ul class="nobullet" style="font-size: 19px;">
<li><i class="fa fa-arrow-circle-right"></i><strong> </strong><strong>Single nucleotide</strong> resolution</li>
<li><i class="fa fa-arrow-circle-right"></i><strong> Gene-specific </strong>and <strong>genome-wide</strong><span> analyses</span></li>
<li><i class="fa fa-arrow-circle-right"></i><strong> NGS</strong><span> </span>compatible</li>
</ul>
<h2>Downstream analysis techniques</h2>
<ul class="square">
<li>Reduced Representation Bisulfite Sequencing (RRBS) with our <a href="https://www.diagenode.com/en/p/premium-rrbs-kit-V2-x24">Premium RRBS Kit V2</a></li>
<li>Bisulfite conversion with our <a href="https://www.diagenode.com/en/p/premium-bisulfite-kit-50-rxns">Premium Bisulfite Kit</a> followed by qPCR, Sanger, Pyrosequencing</li>
</ul>
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<p>The Methylated DNA Immunoprecipitation is based on the affinity purification of methylated and hydroxymethylated DNA using, respectively, an antibody directed against 5-methylcytosine (5-mC) in the case of MeDIP or 5-hydroxymethylcytosine (5-hmC) in the case of hMeDIP.</p>
<h2>How it works</h2>
<p>In brief, Methyl DNA IP is performed as follows: Genomic DNA from cultured cells or tissues is prepared, sheared, and then denatured. Then, immunoselection and immunoprecipitation can take place using the antibody directed against 5 methylcytosine and antibody binding beads. After isolation and purification is performed, the IP’d methylated DNA is ready for any subsequent analysis as qPCR, amplification, hybridization on microarrays or next generation sequencing.</p>
<h2>Applications</h2>
<div align="center"><a href="https://www.diagenode.com/en/p/magmedip-kit-x48-48-rxns" class="center alert radius button"> qPCR analysis</a></div>
<div align="center"><a href="https://www.diagenode.com/en/p/magmedip-kit-x48-48-rxns" class="center alert radius button"> NGS analysis </a></div>
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<li><i class="fa fa-arrow-circle-right"></i> <strong>High enrichment</strong> yield</li>
<li><i class="fa fa-arrow-circle-right"></i> <strong>Robust</strong> & <strong>reproducible</strong> techniques</li>
<li><i class="fa fa-arrow-circle-right"></i> <strong>NGS</strong> compatible</li>
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<li>RRBS_methylated_control.fa: the sequence of the methylated spike-in control in FASTA format</li>
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<p>The DNA methylation control package includes one methylated and one unmethylated spike-in controls together with their corresponding qPCR primer sets for assessing the efficiency of your non-plant MeDIP experiments (Methylated DNA Immunoprecipitation) carried out with Diagenode’s <a href="https://www.diagenode.com/en/p/magmedip-kit-x48-48-rxns">MagMeDIP qPCR Kit</a> and <a href="https://www.diagenode.com/en/p/auto-magmedip-kit-x48-48-rxns" target="_blank">Auto MagMeDIP qPCR Kit</a>.</p>
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<p><em><strong>CAUTION</strong>: These spike-in controls are compatible with <span>Diagenode’s </span><a href="https://www.diagenode.com/en/p/magmedip-kit-x48-48-rxns">MagMeDIP qPCR Kit</a><span><span> </span>and<span> </span></span><a href="https://www.diagenode.com/en/p/auto-magmedip-kit-x48-48-rxns" target="_blank">Auto MagMeDIP qPCR Kit</a><span>. However</span>, those are not the ones directly provided in the kits. The spike-in controls provided in the kits are available separately with the <a href="https://www.diagenode.com/en/p/dna-methylation-control-package-V2-48-rxns">DNA methylation control package V2</a>.</em></p>',
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<li>RRBS_methylated_control.fa: the sequence of the methylated spike-in control in FASTA format</li>
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<h1 class="advertising-feature"><strong>Premium RRBS technology: cost-effective DNA methylation mapping with superior coverage</strong></h1>
<ul class="citation dates">
<li style="text-align: left;" class="vcard last-author"><a href="#auth-5" class="name"><span class="fn">Christoph Bock</span></a><sup><a href="#a2">2</a></sup><sup href="#affil-auth">, </sup></li>
<li style="text-align: left;" class="vcard"><a href="#auth-4" class="name"><span class="fn">Sharon Squazzo</span></a><sup><a href="#a3">3</a></sup><sup>, </sup></li>
<li style="text-align: left;" class="vcard"><a href="#auth-3" class="name"><span class="fn">Miklos Laczik</span></a><sup><a href="#a1">1</a></sup><sup>, </sup></li>
<li style="text-align: left;" class="vcard"><a href="#auth-2" class="name"><span class="fn">Paul Datlinger</span></a><sup><a href="#a2">2</a></sup><sup>, </sup></li>
<li style="text-align: left;" class="vcard c1"><a href="#auth-1" class="name"><span class="fn">Anne-Clémence Veillard</span></a><sup><a href="#a1">1</a></sup><sup>, </sup></li>
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<p>Published online <time datetime="2016-01-28">28 January 2016 </time></p>
<p></p>
<p><a href="http://www.nature.com/nmeth/journal/v13/n2/full/nmeth.f.391.html" class="alert button"><span style="font-weight: 400;">CHECK OUT THE NATURE METHODS PAPER</span></a></p>',
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<li><strong>High throughput</strong> - 96 samples processed in one experiment</li>
<li><strong>Cost-efficient</strong> – Multiplex up to 6 samples/sequencing lane</li>
<li><strong>Validated</strong> with FFPE, cancer, and low-input samples</li>
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<div class="small-6 columns"><br />
<p><b>Superior coverage</b></p>
<p>Comparison of CpG coverage between competing technologies.</p>
<p><strong><em><small>They love it! </small></em></strong><br /><em><small>The new Diagenode Premium RRBS Kit makes it easy to use RRBS cost-effectively and with high throughput, using early sample pooling and multiplex sequencing. Most importantly, the method provides an improved coverage of up to 4 million CpGs for the human genome. We successfully used this protocol on more than 1,000 samples comprising of six different species, various cancers, FFPE and lowinput samples. <br /><strong>Paul Datlinger and Christoph Bock, </strong><strong>CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria </strong></small></em></p>
<p><em> </em></p>
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<div class="row">
<div class="small-6 columns">
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<div class="small-6 columns">
<p><b>Accurate determination of DNA methylation level</b></p>
Excellent results were obtained using Diagenode’s Premium RRBS kit: almost 90% alignment rate, 4.1 million CpGs covered and bisulfite conversion rates around 99.5% for all samples. DNA methylation percentages in the region of IGF2 obtained with the Diagenode’s Premium RRBS Kit. Two human cell lines were analyzed: Gm12878 and MCF7. The MCF7 cell line was studied in duplicates. Each peak represents the DNA methylation percentage at one CpG. The methylated CpGs are shown in red and the unmethylated CpGs in grey.
<p><br /><br /></p>
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<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/landing-pages/rrbs_how_it_works.jpg" alt="rrbs how it works" style="display: block; margin-left: auto; margin-right: auto;" /><br /><br /></p>
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<div class="small-6 columns">
<p><b>How it works</b></p>
By cutting the genome using the <strong>restriction enzyme MspI</strong> (CCGG target sites) followed by size selection, DNA is enriched to represent <strong>CpG-rich genomic regions</strong> (including CpG islands, CpG island shores, enhancers, and other gene-regulatory elements), which are particularly relevant for epigenetic regulation. Similar to exome-sequencing for mutation discovery, the RRBS protocol enriches for some of the most interesting target regions and thereby achieves a reduction in sequencing cost of a factor of 10-20 compared to whole genome bisulfite sequencing.</div>
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<table width="1039">
<tbody>
<tr></tr>
<tr>
<td width="281"> </td>
<td width="379">
<h4>Premium RRBS Kit</h4>
</td>
<td width="379">
<h5>Illumina® RRBS protocol</h5>
</td>
</tr>
<tr>
<td><strong>DNA input</strong></td>
<td>100 ng</td>
<td>2-5 µg</td>
</tr>
<tr>
<td><strong>Multiplexing</strong></td>
<td>Pooling of 6 samples (one HiSeq lane)</td>
<td>no guidelines</td>
</tr>
<tr>
<td><strong>Bisulfite conversion</strong></td>
<td>One bisulfite reaction per pool (6 samples)</td>
<td>One bisulfite reaction per sample</td>
</tr>
<tr>
<td><strong># purification steps</strong></td>
<td>2</td>
<td>7</td>
</tr>
<tr>
<td><strong>Key features</strong></td>
<td width="379">low input - cost-effective - optimized for high-throughput - complete kit</td>
<td width="379">high input - not optimized for high-throughput - reagents from different providers</td>
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<p></p>
<h1 class="advertising-feature"><strong>Premium RRBS technology: cost-effective DNA methylation mapping with superior coverage</strong></h1>
<ul class="citation dates">
<li style="text-align: left;" class="vcard last-author"><a href="#auth-5" class="name"><span class="fn">Christoph Bock</span></a><sup><a href="#a2">2</a></sup><sup href="#affil-auth">, </sup></li>
<li style="text-align: left;" class="vcard"><a href="#auth-4" class="name"><span class="fn">Sharon Squazzo</span></a><sup><a href="#a3">3</a></sup><sup>, </sup></li>
<li style="text-align: left;" class="vcard"><a href="#auth-3" class="name"><span class="fn">Miklos Laczik</span></a><sup><a href="#a1">1</a></sup><sup>, </sup></li>
<li style="text-align: left;" class="vcard"><a href="#auth-2" class="name"><span class="fn">Paul Datlinger</span></a><sup><a href="#a2">2</a></sup><sup>, </sup></li>
<li style="text-align: left;" class="vcard c1"><a href="#auth-1" class="name"><span class="fn">Anne-Clémence Veillard</span></a><sup><a href="#a1">1</a></sup><sup>, </sup></li>
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<p>Nature Methods 13 (2016)</p>
<p>Published online <time datetime="2016-01-28">28 January 2016 </time></p>
<p></p>
<p><a href="http://www.nature.com/nmeth/journal/v13/n2/full/nmeth.f.391.html" class="alert button"><span style="font-weight: 400;">CHECK OUT THE NATURE METHODS PAPER</span></a></p>
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<p> </p>
<div class="small-12 medium-4 large-4 columns"><center></center><center></center><center></center><center><a href="https://www.ncbi.nlm.nih.gov/pubmed/30429608" target="_blank"><img src="https://www.diagenode.com/img/banners/banner-nature-publication-580.png" alt="Click here to read more about MeDIP " caption="false" width="80%" /></a></center></div>
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<h3 style="text-align: justify;">Sensitive tumour detection and classification using plasma cell-free DNA methylomes<br /><a href="https://www.ncbi.nlm.nih.gov/pubmed/30429608" target="_blank">Read the publication</a></h3>
<h3 class="c-article-title u-h1" data-test="article-title" itemprop="name headline" style="text-align: justify;">Preparation of cfMeDIP-seq libraries for methylome profiling of plasma cell-free DNA<br /><a href="https://www.nature.com/articles/s41596-019-0202-2" target="_blank" title="cfMeDIP-seq Nature Method">Read the method</a></h3>
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<p>Perform <strong>MeDIP</strong> (<strong>Me</strong>thylated <strong>D</strong>NA <strong>I</strong>mmuno<strong>p</strong>recipitation) followed by qPCR or NGS to estimate DNA methylation status of your sample using a highly sensitive 5-methylcytosine antibody. Our MagMeDIP kit contains high quality reagents to get the highest enrichment of methylated DNA with an optimized user-friendly protocol.</p>
</div>
</div>
<h3><span>Features</span></h3>
<ul>
<li>Starting DNA amount: <strong>10 ng – 1 µg</strong></li>
<li>Content: <strong>all reagents included</strong> for DNA extraction, immunoprecipitation (including the 5-mC antibody, spike-in controls and their corresponding qPCR primer pairs) as well as DNA isolation after IP.</li>
<li>Application: <strong>qPCR</strong> and <strong>NGS</strong></li>
<li>Robust method, <strong>superior enrichment</strong>, and easy-to-use protocol</li>
<li><strong>High reproducibility</strong> between replicates and repetitive experiments</li>
<li>Compatible with <strong>all species </strong></li>
</ul>',
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<h3><span>How it works</span></h3>
<p>In brief, after the cell collection and lysis, the genomic DNA is extracted, sheared, and then denatured. In the next step the antibody directed against 5 methylcytosine and antibody binding beads are used for immunoselection and immunoprecipitation of methylated DNA fragments. Then, the IP’d methylated DNA is isolated and can be used for any subsequent analysis as qPCR, amplification, hybridization on microarrays or next generation sequencing.</p>
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'info2' => '<p>The kit MagMeDIP contains all reagents necessary for a complete MeDIP-qPCR workflow. Two MagMeDIP protocols have been validated: for manual processing as well as for automated processing, using the Diagenode’s IP-Star Compact Automated System (please refer to the kit manual).</p>
<ul>
<li><strong>Complete kit</strong> including DNA extraction module, IP antibody and reagents, DNA isolation buffer</li>
<li><strong>Quality control of the IP:</strong> due to methylated and unmethylated DNA spike-in controls and their associated qPCR primers</li>
<li><strong>Easy to use</strong> with user-friendly magnetic beads and rack</li>
<li><strong>Highly validated protocol</strong></li>
<li>Automated protocol supplied</li>
</ul>
<center><img src="https://www.diagenode.com/img/product/kits/fig1-magmedipkit.png" width="85%" alt="Methylated DNA Immunoprecipitation" caption="false" /></center>
<p style="font-size: 0.9em;"><em><strong>Figure 1.</strong> Immunoprecipitation results obtained with Diagenode MagMeDIP Kit</em></p>
<p style="font-size: 0.9em;">MeDIP assays were performed manually using 1 µg or 50 ng gDNA from blood cells with the MagMeDIP kit (Diagenode). The IP was performed with the Methylated and Unmethylated spike-in controls included in the kit, together with the human DNA samples. The DNA was isolated/purified using DIB. Afterwards, qPCR was performed using the primer pairs included in this kit.</p>
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'info3' => '<p>For DNA methylation analysis on the whole genome, MagMeDIP kit can be coupled with Next-Generation Sequencing. To perform MeDIP-sequencing we recommend the following strategy:</p>
<ul style="list-style-type: circle;">
<li>Choose a library preparation solution which is compatible with the starting amount of DNA you are planning to use (from 10 ng to 1 μg). It can be a home-made solution or a commercial one.</li>
<li>Choose the indexing system that fits your needs considering the following features:</li>
<ul>
<ul>
<ul>
<li>Single-indexing, combinatorial dual-indexing or unique dual-indexing</li>
<li>Number of barcodes</li>
<li>Full-length adaptors containing the barcodes or barcoding at the final amplification step</li>
<li>Presence / absence of Unique Molecular Identifiers (for PCR duplicates removal)</li>
</ul>
</ul>
</ul>
<li>Standard library preparation protocols are compatible with double-stranded DNA only, therefore the first steps of the library preparation (end repair, A-tailing, adaptor ligation and clean-up) will have to be performed on sheared DNA, before the IP.</li>
</ul>
<p style="padding-left: 30px;"><strong>CAUTION:</strong> As the immunoprecipitation step occurs at the middle of the library preparation workflow, single-tube solutions for library preparation are usually not compatible with MeDIP-sequencing.</p>
<ul style="list-style-type: circle;">
<li>For DNA isolation after the IP, we recommend using the <a href="https://www.diagenode.com/en/p/ipure-kit-v2-x24" title="IPure kit v2">IPure kit v2</a> (available separately, Cat. No. C03010014) instead of DNA isolation Buffer.</li>
</ul>
<ul style="list-style-type: circle;">
<li>Perform library amplification after the DNA isolation following the standard protocol of the chosen library preparation solution.</li>
</ul>
<h3><span>MeDIP-seq workflow</span></h3>
<center><img src="https://www.diagenode.com/img/product/kits/MeDIP-seq-workflow.png" width="110%" alt="MagMeDIP qPCR Kit x10 workflow" caption="false" /></center>
<h3><span>Example of results</span></h3>
<center><img src="https://www.diagenode.com/img/product/kits/medip-specificity.png" alt="MagMeDIP qPCR Kit Result" caption="false" width="951" height="488" /></center>
<p></p>
<p style="font-size: 0.9em;"><strong>Figure 1. qPCR analysis of external spike-in DNA controls (methylated and unmethylated) after IP.</strong> Samples were prepared using 1μg – 100ng -10ng sheared human gDNA with the MagMeDIP kit (Diagenode) and a commercially available library prep kit. DNA isolation after IP has been performed with IPure kit V2 (Diagenode).</p>
<p></p>
<p></p>
<center><img src="https://www.diagenode.com/img/product/kits/medip-saturation-analysis.png" alt=" MagMeDIP kit " caption="false" width="951" height="461" /></center>
<p></p>
<p style="font-size: 0.9em;"><strong>Figure 2. Saturation analysis.</strong> Clean reads were aligned to the human genome (hg19) using Burrows-Wheeler aligner (BWA) algorithm after which duplicated and unmapped reads were removed resulting in a mapping efficiency >98% for all samples. Quality and validity check of the mapped MeDIP-seq data was performed using MEDIPS R package. Saturation plots show that all sets of reads have sufficient complexity and depth to saturate the coverage profile of the reference genome and that this is reproducible between replicates and repetitive experiments (data shown for 50 ng gDNA input: left panel = replicate a, right panel = replicate b).</p>
<p></p>
<p></p>
<center><img src="https://www.diagenode.com/img/product/kits/medip-libraries-prep.png" alt="MagMeDIP x10 " caption="false" width="951" height="708" /></center>
<p></p>
<p style="font-size: 0.9em;"><strong>Figure 3. Sequencing profiles of MeDIP-seq libraries prepared from different starting amounts of sheared gDNA on the positive and negative methylated control regions.</strong> MeDIP-seq libraries were prepared from decreasing starting amounts of gDNA (1 μg (green), 50 ng (red), and 10ng (blue)) originating from human blood with the MagMeDIP kit (Diagenode) and a commercially available library prep kit. DNA isolation after IP has been performed with IPure kit V2 (Diagenode). IP and corresponding INPUT samples were sequenced on Illumina NovaSeq SP with 2x50 PE reads. The reads were mapped to the human genome (hg19) with bwa and the alignments were loaded into IGV (the tracks use an identical scale). The top IGV figure shows the TSH2B (also known as H2BC1) gene (marked by blue boxes in the bottom track) and its surroundings. The TSH2B gene is coding for a histone variant that does not occur in blood cells, and it is known to be silenced by methylation. Accordingly, we see a high coverage in the vicinity of this gene. The bottom IGV figure shows the GADPH locus (marked by blue boxes in the bottom track) and its surroundings. The GADPH gene is a highly active transcription region and should not be methylated, resulting in no reads accumulation following MeDIP-seq experiment.</p>
<p></p>
<ul>
<ul>
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'meta_description' => 'Perform Methylated DNA Immunoprecipitation (MeDIP) to estimate DNA methylation status of your sample using highly specific 5-mC antibody. This kit allows the preparation of cfMeDIP-seq libraries.',
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'description' => '<p><a href="https://www.diagenode.com/files/products/kits/premium-RRBS-spike-in%20controls-08_18.pdf"><img src="https://www.diagenode.com/img/buttons/bt-manual.png" /></a></p>
<p>The DNA methylation control package includes one methylated and one unmethylated spike-in controls together with their corresponding qPCR primer sets for assessing the efficiency of your non-plant MeDIP experiments (Methylated DNA Immunoprecipitation) carried out with Diagenode’s <a href="https://www.diagenode.com/en/p/magmedip-kit-x48-48-rxns">MagMeDIP qPCR Kit</a> and <a href="https://www.diagenode.com/en/p/auto-magmedip-kit-x48-48-rxns" target="_blank">Auto MagMeDIP qPCR Kit</a>.</p>
<p><span>Those spike-in controls are made from <em>A. thaliana</em><span>. Therefore, they can interfere with DNA samples derived from plant species.</span></span></p>
<p><em><strong>CAUTION</strong>: These spike-in controls are compatible with <span>Diagenode’s </span><a href="https://www.diagenode.com/en/p/magmedip-kit-x48-48-rxns">MagMeDIP qPCR Kit</a><span><span> </span>and<span> </span></span><a href="https://www.diagenode.com/en/p/auto-magmedip-kit-x48-48-rxns" target="_blank">Auto MagMeDIP qPCR Kit</a><span>. However</span>, those are not the ones directly provided in the kits. The spike-in controls provided in the kits are available separately with the <a href="https://www.diagenode.com/en/p/dna-methylation-control-package-V2-48-rxns">DNA methylation control package V2</a>.</em></p>',
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'meta_description' => 'DNA Methylation control package',
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'description' => '<div class="row">
<div class="large-12 columns">
<div style="text-align: justify;" class="small-12 medium-8 large-8 columns">
<h2>Complete solutions for DNA methylation studies</h2>
<p>Whether you are experienced or new to the field of DNA methylation, Diagenode has everything you need to make your assay as easy and convenient as possible while ensuring consistent data between samples and experiments. Diagenode offers sonication instruments, reagent kits, high quality antibodies, and high-throughput automation capability to address all of your specific DNA methylation analysis requirements.</p>
</div>
<div class="small-12 medium-4 large-4 columns text-center"><a href="../landing-pages/dna-methylation-grant-applications"><img src="https://www.diagenode.com/img/banners/banner-dna-grant.png" alt="" /></a></div>
<div style="text-align: justify;" class="small-12 medium-12 large-12 columns">
<p>DNA methylation was the first discovered epigenetic mark and is the most widely studied topic in epigenetics. <em>In vivo</em>, DNA is methylated following DNA replication and is involved in a number of biological processes including the regulation of imprinted genes, X chromosome inactivation. and tumor suppressor gene silencing in cancer cells. Methylation often occurs in cytosine-guanine rich regions of DNA (CpG islands), which are commonly upstream of promoter regions.</p>
</div>
<div class="small-12 medium-12 large-12 columns"><br /><br />
<ul class="accordion" data-accordion="">
<li class="accordion-navigation"><a href="#dnamethyl"><i class="fa fa-caret-right"></i> Learn more</a>
<div id="dnamethyl" class="content">5-methylcytosine (5-mC) has been known for a long time as the only modification of DNA for epigenetic regulation. In 2009, however, Kriaucionis discovered a second methylated cytosine, 5-hydroxymethylcytosine (5-hmC). The so-called 6th base, is generated by enzymatic conversion of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine by the TET family of oxygenases. Early reports suggested that 5-hmC may represent an intermediate of active demethylation in a new pathway which demethylates DNA, converting 5-mC to cytosine. Recent evidence fuel this hypothesis suggesting that further oxidation of the hydroxymethyl group leads to a formyl or carboxyl group followed by either deformylation or decarboxylation. The formyl and carboxyl groups of 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) could be enzymatically removed without excision of the base.
<p class="text-center"><img src="https://www.diagenode.com/img/categories/kits_dna/dna_methylation_variants.jpg" /></p>
</div>
</li>
</ul>
<br />
<h2>Main DNA methylation technologies</h2>
<p style="text-align: justify;">Overview of the <span style="font-weight: 400;">three main approaches for studying DNA methylation.</span></p>
<div class="row">
<ol>
<li style="font-weight: 400;"><span style="font-weight: 400;">Chemical modification with bisulfite – Bisulfite conversion</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Enrichment of methylated DNA (including MeDIP and MBD)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Treatment with methylation-sensitive or dependent restriction enzymes</span></li>
</ol>
<p><span style="font-weight: 400;"> </span></p>
<div class="row">
<table>
<thead>
<tr>
<th></th>
<th>Description</th>
<th width="350">Features</th>
</tr>
</thead>
<tbody>
<tr>
<td><strong>Bisulfite conversion</strong></td>
<td><span style="font-weight: 400;">Chemical conversion of unmethylated cytosine to uracil. Methylated cytosines are protected from this conversion allowing to determine DNA methylation at single nucleotide resolution.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Single nucleotide resolution</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Quantitative analysis - methylation rate (%)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Gold standard and well studied</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Compatible with automation</span></li>
</ul>
</td>
</tr>
<tr>
<td><b>Methylated DNA enrichment</b></td>
<td><span style="font-weight: 400;">(Hydroxy-)Methylated DNA is enriched by using specific antibodies (hMeDIP or MeDIP) or proteins (MBD) that specifically bind methylated CpG sites in fragmented genomic DNA.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Resolution depends on the fragment size of the enriched methylated DNA (300 bp)</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Qualitative analysis</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Compatible with automation</span></li>
</ul>
</td>
</tr>
<tr>
<td><strong>Restriction enzyme-based digestion</strong></td>
<td><span style="font-weight: 400;">Use of (hydroxy)methylation-sensitive or (hydroxy)methylation-dependent restriction enzymes for DNA methylation analysis at specific sites.</span></td>
<td>
<ul style="list-style-type: circle;">
<li style="font-weight: 400;"><span style="font-weight: 400;">Determination of methylation status is limited by the enzyme recognition site</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Easy to use</span></li>
</ul>
</td>
</tr>
</tbody>
</table>
</div>
</div>
<div class="row"></div>
</div>
</div>
<div class="large-12 columns"></div>
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'meta_description' => 'Complete, optimized solutions for analyzing DNA methylation manually or on our automated system.',
'meta_title' => 'DNA Methylation - Bisulfite sequencing - Epigenetics | Diagenode',
'modified' => '2019-03-25 10:07:27',
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<div style="text-align: justify;" class="large-12 columns">Bisulfite modification of DNA is the most commonly used, "<strong>gold standard</strong>" method for DNA methylation studies providing <strong>single nucleotide resolution</strong>. T<span style="font-weight: 400;">his technology is based on the chemical conversion of unmethylated cytosine to uracil. Methylated cytosines are protected from this conversion allowing to determine DNA methylation at the singe nucleotide level.</span></div>
<div style="text-align: justify;" class="large-12 columns"></div>
<div style="text-align: justify;" class="large-12 columns">Various analyses can be performed on the altered sequence to retrieve this information: bisulfite sequencing, pyrosequencing, methylation-specific PCR, high resolution melting curve analysis, microarray-based approaches, and next-generation sequencing.
<h3>How it works</h3>
Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected (see Figure 1).
<p class="text-center"><img src="https://www.diagenode.com/img/applications/bisulfite.png" /><br />Figure 1: Overview of bisulfite conversion of DNA</p>
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'meta_title' => 'Bisulfite sequencing(Bis-Seq) - Bisulfite conversion - DNA Methylation | Diagenode',
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<div class="small-12 medium-3 large-3 columns"><center><a href="https://www.ncbi.nlm.nih.gov/pubmed/30429608" target="_blank"><img src="https://www.diagenode.com/img/banners/banner-nature-publication-580.png" /></a></center></div>
<div class="small-12 medium-9 large-9 columns">
<h3>Sensitive tumour detection and classification using plasma cell-free DNA methylomes<br /><a href="https://www.ncbi.nlm.nih.gov/pubmed/30429608" target="_blank">Read the publication</a></h3>
<h3 class="c-article-title u-h1" data-test="article-title" itemprop="name headline">Preparation of cfMeDIP-seq libraries for methylome profiling of plasma cell-free DNA<br /><a href="https://www.nature.com/articles/s41596-019-0202-2" target="_blank" title="cfMeDIP-seq Nature Method">Read the method</a></h3>
</div>
</div>
<div class="row">
<div class="large-12 columns"><span>The Methylated DNA Immunoprecipitation is based on the affinity purification of methylated and hydroxymethylated DNA using, respectively, an antibody directed against 5-methylcytosine (5-mC) in the case of MeDIP or 5-hydroxymethylcytosine (5-hmC) in the case of hMeDIP.</span><br />
<h2></h2>
<h2>How it works</h2>
<p>In brief, Methyl DNA IP is performed as follows: Genomic DNA from cultured cells or tissues is prepared, sheared, and then denatured. Then, immunoselection and immunoprecipitation can take place using the antibody directed against 5 methylcytosine and antibody binding beads. After isolation and purification is performed, the IP’d methylated DNA is ready for any subsequent analysis as qPCR, amplification, hybridization on microarrays or next generation sequencing.</p>
<h2>Applications</h2>
<div align="center"><a href="https://www.diagenode.com/en/p/magmedip-kit-x48-48-rxns" class="center alert radius button"> qPCR analysis</a></div>
<div align="center"><a href="https://www.diagenode.com/en/p/magmedip-seq-package-V2-x10" class="center alert radius button"> NGS analysis </a></div>
<h2>Advantages</h2>
<ul style="font-size: 19px;" class="nobullet">
<li><i class="fa fa-arrow-circle-right"></i> <strong>Unaffected</strong> DNA</li>
<li><i class="fa fa-arrow-circle-right"></i> <strong>High enrichment</strong> yield</li>
<li><i class="fa fa-arrow-circle-right"></i> <strong>Robust</strong> & <strong>reproducible</strong> techniques</li>
<li><i class="fa fa-arrow-circle-right"></i> <strong>NGS</strong> compatible</li>
</ul>
<h2></h2>
</div>
</div>
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<p>Sodium bisulfite conversion of genomic DNA is the most commonly used method for DNA methylation studies providing <strong>single nucleotide resolution</strong>. It enables <span>to differentiate and detect unmethylated versus methylated cytosines. This procedure can then be followed either by <strong>PCR amplification</strong> or <strong>next generation sequencing</strong> to reveal the methylation status of every cytosine in gene specific amplification or whole genome amplification.</span></p>
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<h2>How it works</h2>
<p style="text-align: left;">Treatment of DNA with sodium bisulfite converts unmethylated cytosine to uracil, while methylated cytosines remain unchanged. <span>The DNA is then amplified by PCR where the uracils are converted to thymines. </span></p>
<p style="text-align: center;"><span></span></p>
<p><img src="https://www.diagenode.com/img/categories/bisulfite-conversion/bisulfite-conversion-acgautac.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<h2>Advantages</h2>
<ul class="nobullet" style="font-size: 19px;">
<li><i class="fa fa-arrow-circle-right"></i><strong> </strong><strong>Single nucleotide</strong> resolution</li>
<li><i class="fa fa-arrow-circle-right"></i><strong> Gene-specific </strong>and <strong>genome-wide</strong><span> analyses</span></li>
<li><i class="fa fa-arrow-circle-right"></i><strong> NGS</strong><span> </span>compatible</li>
</ul>
<h2>Downstream analysis techniques</h2>
<ul class="square">
<li>Reduced Representation Bisulfite Sequencing (RRBS) with our <a href="https://www.diagenode.com/en/p/premium-rrbs-kit-V2-x24">Premium RRBS Kit V2</a></li>
<li>Bisulfite conversion with our <a href="https://www.diagenode.com/en/p/premium-bisulfite-kit-50-rxns">Premium Bisulfite Kit</a> followed by qPCR, Sanger, Pyrosequencing</li>
</ul>
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<p>The Methylated DNA Immunoprecipitation is based on the affinity purification of methylated and hydroxymethylated DNA using, respectively, an antibody directed against 5-methylcytosine (5-mC) in the case of MeDIP or 5-hydroxymethylcytosine (5-hmC) in the case of hMeDIP.</p>
<h2>How it works</h2>
<p>In brief, Methyl DNA IP is performed as follows: Genomic DNA from cultured cells or tissues is prepared, sheared, and then denatured. Then, immunoselection and immunoprecipitation can take place using the antibody directed against 5 methylcytosine and antibody binding beads. After isolation and purification is performed, the IP’d methylated DNA is ready for any subsequent analysis as qPCR, amplification, hybridization on microarrays or next generation sequencing.</p>
<h2>Applications</h2>
<div align="center"><a href="https://www.diagenode.com/en/p/magmedip-kit-x48-48-rxns" class="center alert radius button"> qPCR analysis</a></div>
<div align="center"><a href="https://www.diagenode.com/en/p/magmedip-kit-x48-48-rxns" class="center alert radius button"> NGS analysis </a></div>
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<li><i class="fa fa-arrow-circle-right"></i> <strong>High enrichment</strong> yield</li>
<li><i class="fa fa-arrow-circle-right"></i> <strong>Robust</strong> & <strong>reproducible</strong> techniques</li>
<li><i class="fa fa-arrow-circle-right"></i> <strong>NGS</strong> compatible</li>
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<li>RRBS_methylated_control.fa: the sequence of the methylated spike-in control in FASTA format</li>
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<p>The DNA methylation control package includes one methylated and one unmethylated spike-in controls together with their corresponding qPCR primer sets for assessing the efficiency of your non-plant MeDIP experiments (Methylated DNA Immunoprecipitation) carried out with Diagenode’s <a href="https://www.diagenode.com/en/p/magmedip-kit-x48-48-rxns">MagMeDIP qPCR Kit</a> and <a href="https://www.diagenode.com/en/p/auto-magmedip-kit-x48-48-rxns" target="_blank">Auto MagMeDIP qPCR Kit</a>.</p>
<p><span>Those spike-in controls are made from <em>A. thaliana</em><span>. Therefore, they can interfere with DNA samples derived from plant species.</span></span></p>
<p><em><strong>CAUTION</strong>: These spike-in controls are compatible with <span>Diagenode’s </span><a href="https://www.diagenode.com/en/p/magmedip-kit-x48-48-rxns">MagMeDIP qPCR Kit</a><span><span> </span>and<span> </span></span><a href="https://www.diagenode.com/en/p/auto-magmedip-kit-x48-48-rxns" target="_blank">Auto MagMeDIP qPCR Kit</a><span>. However</span>, those are not the ones directly provided in the kits. The spike-in controls provided in the kits are available separately with the <a href="https://www.diagenode.com/en/p/dna-methylation-control-package-V2-48-rxns">DNA methylation control package V2</a>.</em></p>',
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<li>RRBS_methylated_control.fa: the sequence of the methylated spike-in control in FASTA format</li>
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×
