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<h1 class="advertising-feature"><strong>Premium RRBS technology: cost-effective DNA methylation mapping with superior coverage</strong></h1>
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<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>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>
<li><strong>High efficiency and minimal bias</strong> - Low DNA degradation and reduced amplification</li>
<li><strong>Complete kit</strong> – Bisulfite conversion reagents, MspI enzyme, library preparation reagents including barcodes, and spike-in controls</li>
<li class="accordion-navigation" style="list-style-type: circle; display: list-item;"><strong>Already tested on various species </strong>– human, mouse, rat, pig, cow, dog, zebrafish, Daphnia <a href="#species" style="color: #13b29c; background-color: transparent; display: inline; padding: 0;">and more</a> <span class="content" id="species">cichlid fish (Astatotilapia calliptera), mossy frog, yellow-bellied slider, dice snake, zebra finch, humboldt penguin, leaf bird, buzzard, vulturine guinea fowe, parma kangaroo, cheetah, mouflon</span></li>
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<p> </p>
<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/kits/rrbs-figure-1.png" alt="Chr Shearing" style="display: block; margin-left: auto; margin-right: auto;" /></p>
</div>
<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">
<p><img src="https://www.diagenode.com/img/product/kits/rrbs-figure-2.jpg" alt="dna methylation" style="display: block; margin-left: auto; margin-right: auto;" /></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>
</div>
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<div class="row">
<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>
</div>
<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>
</div>
<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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<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>
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<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>
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<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>
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<li style="font-weight: 400;"><span style="font-weight: 400;">Determination of methylation status is limited by the enzyme recognition site</span></li>
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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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<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>
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<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>
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<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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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.
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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">
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<h4>Premium RRBS Kit</h4>
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<td width="379">
<h5>Illumina® RRBS protocol</h5>
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<td>100 ng</td>
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<td>no guidelines</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>
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<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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<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>
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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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<li><strong>Complete kit</strong> – Bisulfite conversion reagents, MspI enzyme, library preparation reagents including barcodes, and spike-in controls</li>
<li class="accordion-navigation" style="list-style-type: circle; display: list-item;"><strong>Already tested on various species </strong>– human, mouse, rat, pig, cow, dog, zebrafish, Daphnia <a href="#species" style="color: #13b29c; background-color: transparent; display: inline; padding: 0;">and more</a> <span class="content" id="species">cichlid fish (Astatotilapia calliptera), mossy frog, yellow-bellied slider, dice snake, zebra finch, humboldt penguin, leaf bird, buzzard, vulturine guinea fowe, parma kangaroo, cheetah, mouflon</span></li>
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<div class="row">
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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">
<p><img src="https://www.diagenode.com/img/product/kits/rrbs-figure-2.jpg" alt="dna methylation" style="display: block; margin-left: auto; margin-right: auto;" /></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>
</div>
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<div class="row">
<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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<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>
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<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">
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<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>
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<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>
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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>
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<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>
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<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>
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<li><strong>Superior coverage</strong> – 4 million CpGs</li>
<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>
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<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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<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.
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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">
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<td width="281"> </td>
<td width="379">
<h4>Premium RRBS Kit</h4>
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<td width="379">
<h5>Illumina® RRBS protocol</h5>
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<td><strong>DNA input</strong></td>
<td>100 ng</td>
<td>2-5 µg</td>
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<td><strong>Multiplexing</strong></td>
<td>Pooling of 6 samples (one HiSeq lane)</td>
<td>no guidelines</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>
</ul>
<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>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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<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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<p><br /><br /></p>
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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>
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<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>
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<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>
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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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<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>
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<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>
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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>
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<li><i class="fa fa-arrow-circle-right"></i><strong> NGS</strong><span> </span>compatible</li>
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<h2>Downstream analysis techniques</h2>
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<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>
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<li><strong>Superior coverage</strong> – 4 million CpGs</li>
<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>
<li><strong>High efficiency and minimal bias</strong> - Low DNA degradation and reduced amplification</li>
<li><strong>Complete kit</strong> – Bisulfite conversion reagents, MspI enzyme, library preparation reagents including barcodes, and spike-in controls</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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<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.
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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">
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<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>
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<td><strong>DNA input</strong></td>
<td>100 ng</td>
<td>2-5 µg</td>
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<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>
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<td><strong># purification steps</strong></td>
<td>2</td>
<td>7</td>
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<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>
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<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>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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<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>
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<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>Superior coverage</strong> – 4 million CpGs</li>
<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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<li class="accordion-navigation" style="list-style-type: circle; display: list-item;"><strong>Already tested on various species </strong>– human, mouse, rat, pig, cow, dog, zebrafish, Daphnia <a href="#species" style="color: #13b29c; background-color: transparent; display: inline; padding: 0;">and more</a> <span class="content" id="species">cichlid fish (Astatotilapia calliptera), mossy frog, yellow-bellied slider, dice snake, zebra finch, humboldt penguin, leaf bird, buzzard, vulturine guinea fowe, parma kangaroo, cheetah, mouflon</span></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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<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>
</tr>
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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>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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<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>
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<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>
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<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.
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<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>
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<thead>
<tr>
<th></th>
<th>Description</th>
<th width="350">Features</th>
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<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>
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<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>
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</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>
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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>
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<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>
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<h2>Downstream analysis techniques</h2>
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<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>
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<li><strong>Cost-efficient</strong> – Multiplex up to 6 samples/sequencing lane</li>
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<li><strong>High efficiency and minimal bias</strong> - Low DNA degradation and reduced amplification</li>
<li><strong>Complete kit</strong> – Bisulfite conversion reagents, MspI enzyme, library preparation reagents including barcodes, and spike-in controls</li>
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<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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<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.
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<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>
</div>
<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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<tbody>
<tr></tr>
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<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>
</tr>
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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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