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<p class="text-center"><img src="https://www.diagenode.com/img/product/antibodies/C15200081_ChIPSeq-A.png" alt="5-mC (5-methylcytosine) Antibody validated in MeDIP-seq" caption="false" width="886" height="173" /></p>
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<p><small><strong>Figure 1. MeDIP-seq with the Diagenode monoclonal antibody directed against 5-mC</strong><br /> Genomic DNA from E14 ES cells was sheared with the Bioruptor® to generate random fragments (size range 300 to 700 bp). One µg of the fragmented DNA was ligated to Illumina adapters and the resulting DNA was used for a standard MeDIP assay, using 2 µg of the Diagenode monoclonal against 5-mC (Cat. No. C15200081). After recovery of the methylated DNA, Illumina sequencing libraries were generated and sequenced on an Illumina Genome Analyzer according to the manufacturer’s instructions. Figure 1A and 1B show Genome browser views of CA simple repeat elements with read distributions specific for 5-mC at 2 gene locations (SigleC15 and Mfsd4). Visual inspection of the peak profiles in a genome browser reveals high enrichment of CA simple repeats in affinity-enriched methylated fragments after MeDIP with the Diagenode 5-mC monoclonal antibody.</small></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15200081_medip.png" alt="5-mC (5-methylcytosine) Antibody validated in MeDIP" caption="false" width="355" height="372" /></p>
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<p><small><strong>Figure 2. MeDIP results obtained with the Diagenode monoclonal antibody directed against 5-mC</strong><br /> MeDIP (Methylated DNA immunoprecipitation) was performed on 1 µg fragmented human genomic DNA using 0.2 µg of the Diagenode monoclonal antibody against 5-mC (cat. No. C15200081) and the MagMeDIP Kit (cat. No. C02010021). The fragmented DNA was spiked with the internal controls present in the kit (methylated DNA (meDNA) as a positive and unmethylated DNA (unDNA) as a negative control) prior to performing the IP. QPCR was performed with optimized primer sets, included in the kit, specific for the methylated and unmethylated DNA controls, and for a known methylated (TSH2B) and unmethylated (GAPDH) genomic region. Figure 2 shows the recovery expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).</small></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15200081_Dotblot.png" alt=" 5-mC (5-methylcytosine) Antibody validated in dot blot" caption="false" width="201" height="196" /></p>
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<p><small><strong>Figure 3. Dot blot analysis using the Diagenode monoclonal antibody directed against 5-mC</strong><br />To demonstrate the specificity of the Diagenode antibody against 5-mC (cat. No. C15200081), a Dot blot analysis was performed using the hmC, mC and C controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (cat. No. C02040010). One hundred to 4 ng (equivalent of 5 to 0.2 pmol of C-bases) of the controls were spotted on a membrane. Figure 3 shows a high specificity of the antibody for the methylated control.</small></p>
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<div class="small-12 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15200081_IF1.png" alt="5-mC (5-methylcytosine) Antibody for immunofluorescence" height="121" width="500" caption="false" /></center></div>
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<p><small><strong>Figure 4. Immunofluorescence using the Diagenode monoclonal antibody directed against 5-mC</strong><br />HeLa cells were stained with the Diagenode antibody against 5-mC (Cat. No. C15200081) and with DAPI. Cells were fixed with 4% formaldehyde for 10’ and blocked with PBS/TX-100 containing 1% BSA. The cells were immunofluorescently labelled with the 5-mC antibody (middle) diluted 1:500 in blocking solution followed by an anti-mouse antibody conjugated to Alexa594. The left panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.</small></p>
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<!--
<div class="row">
<div class="small-12 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15200081_SPR.png" alt="5-methylcytosine (5-mC) Antibody" surface="" plasmon="" resonance="" caption="false" width="700" height="372" /></center></div>
</div>
<div class="row">
<div class="small-12 columns">
<p><small><strong>Figure 5. Surface plasmon resonance (SPR) analysis of the the Diagenode monoclonal antibody directed against 5-mC</strong><br />A synthesized biotin-labeled 5-mC conjugate was immobilized on a CM4 BIAcore sensorchip (GE Healthcare, France). Briefly, two flowcells were prepared by sequential injections of EDC/NHS, streptavidin, and ethanolamine. One of these flowcells served as negative control (biotinylated spacer without 5-mC), while biotinylated 5-mC conjugate was injected in the other one, to get an immobilization level of 55 response units (RU). All SPR experiments were performed, using HBS-N buffer (10 mM HEPES,150 mM NaCl, pH 7.4), at a flow rate of 5 µl/min. Interaction assays involved injections of 2 different dilutions of the Diagenode 5-mC monoclonal antibody (Cat. No. C15200081) over the biotinylated 5-mC conjugate and negative control surfaces, followed by a 3 min washing step with HBS-N buffer to allow dissociation of the complexes formed. At the end of each cycle, the streptavidin surface was regenerated by injection of 0.1M citric acid (pH=3).</small></p>
<p><small>The sensorgrams correspond to the biotinylated 5-mC conjugate surface signal subtracted with the negative control. Data from the sensorgrams that reached binding equilibrium were used for Scatchard analysis. The value of the dissociation constant (kd) obtained by global fitting and 1:1 Langmuir model is 65 nM.</small></p>
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<td>Fig 1, 2</td>
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<p><small><strong>Figure 1. MeDIP-seq with the Diagenode monoclonal antibody directed against 5-mC</strong><br /> Genomic DNA from E14 ES cells was sheared with the Bioruptor® to generate random fragments (size range 300 to 700 bp). One µg of the fragmented DNA was ligated to Illumina adapters and the resulting DNA was used for a standard MeDIP assay, using 2 µg of the Diagenode monoclonal against 5-mC (Cat. No. C15200081). After recovery of the methylated DNA, Illumina sequencing libraries were generated and sequenced on an Illumina Genome Analyzer according to the manufacturer’s instructions. Figure 1A and 1B show Genome browser views of CA simple repeat elements with read distributions specific for 5-mC at 2 gene locations (SigleC15 and Mfsd4). Visual inspection of the peak profiles in a genome browser reveals high enrichment of CA simple repeats in affinity-enriched methylated fragments after MeDIP with the Diagenode 5-mC monoclonal antibody.</small></p>
</div>
</div>
<div class="row">
<div class="small-5 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15200081_medip.png" alt="5-mC (5-methylcytosine) Antibody validated in MeDIP" caption="false" width="355" height="372" /></p>
</div>
<div class="small-7 columns">
<p><small><strong>Figure 2. MeDIP results obtained with the Diagenode monoclonal antibody directed against 5-mC</strong><br /> MeDIP (Methylated DNA immunoprecipitation) was performed on 1 µg fragmented human genomic DNA using 0.2 µg of the Diagenode monoclonal antibody against 5-mC (cat. No. C15200081) and the MagMeDIP Kit (cat. No. C02010021). The fragmented DNA was spiked with the internal controls present in the kit (methylated DNA (meDNA) as a positive and unmethylated DNA (unDNA) as a negative control) prior to performing the IP. QPCR was performed with optimized primer sets, included in the kit, specific for the methylated and unmethylated DNA controls, and for a known methylated (TSH2B) and unmethylated (GAPDH) genomic region. Figure 2 shows the recovery expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).</small></p>
</div>
</div>
<div class="row">
<div class="small-3 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15200081_Dotblot.png" alt=" 5-mC (5-methylcytosine) Antibody validated in dot blot" caption="false" width="201" height="196" /></p>
</div>
<div class="small-9 columns">
<p><small><strong>Figure 3. Dot blot analysis using the Diagenode monoclonal antibody directed against 5-mC</strong><br />To demonstrate the specificity of the Diagenode antibody against 5-mC (cat. No. C15200081), a Dot blot analysis was performed using the hmC, mC and C controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (cat. No. C02040010). One hundred to 4 ng (equivalent of 5 to 0.2 pmol of C-bases) of the controls were spotted on a membrane. Figure 3 shows a high specificity of the antibody for the methylated control.</small></p>
</div>
</div>
<div class="row">
<div class="small-12 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15200081_IF1.png" alt="5-mC (5-methylcytosine) Antibody for immunofluorescence" height="121" width="500" caption="false" /></center></div>
</div>
<div class="row">
<div class="small-12 columns">
<p><small><strong>Figure 4. Immunofluorescence using the Diagenode monoclonal antibody directed against 5-mC</strong><br />HeLa cells were stained with the Diagenode antibody against 5-mC (Cat. No. C15200081) and with DAPI. Cells were fixed with 4% formaldehyde for 10’ and blocked with PBS/TX-100 containing 1% BSA. The cells were immunofluorescently labelled with the 5-mC antibody (middle) diluted 1:500 in blocking solution followed by an anti-mouse antibody conjugated to Alexa594. The left panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.</small></p>
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<!--
<div class="row">
<div class="small-12 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15200081_SPR.png" alt="5-methylcytosine (5-mC) Antibody" surface="" plasmon="" resonance="" caption="false" width="700" height="372" /></center></div>
</div>
<div class="row">
<div class="small-12 columns">
<p><small><strong>Figure 5. Surface plasmon resonance (SPR) analysis of the the Diagenode monoclonal antibody directed against 5-mC</strong><br />A synthesized biotin-labeled 5-mC conjugate was immobilized on a CM4 BIAcore sensorchip (GE Healthcare, France). Briefly, two flowcells were prepared by sequential injections of EDC/NHS, streptavidin, and ethanolamine. One of these flowcells served as negative control (biotinylated spacer without 5-mC), while biotinylated 5-mC conjugate was injected in the other one, to get an immobilization level of 55 response units (RU). All SPR experiments were performed, using HBS-N buffer (10 mM HEPES,150 mM NaCl, pH 7.4), at a flow rate of 5 µl/min. Interaction assays involved injections of 2 different dilutions of the Diagenode 5-mC monoclonal antibody (Cat. No. C15200081) over the biotinylated 5-mC conjugate and negative control surfaces, followed by a 3 min washing step with HBS-N buffer to allow dissociation of the complexes formed. At the end of each cycle, the streptavidin surface was regenerated by injection of 0.1M citric acid (pH=3).</small></p>
<p><small>The sensorgrams correspond to the biotinylated 5-mC conjugate surface signal subtracted with the negative control. Data from the sensorgrams that reached binding equilibrium were used for Scatchard analysis. The value of the dissociation constant (kd) obtained by global fitting and 1:1 Langmuir model is 65 nM.</small></p>
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'slug' => '5-mc-monoclonal-antibody-33d3-premium-100-ug-50-ul',
'meta_title' => '5-methylcytosine (5-mC) Antibody - clone 33D3 (C15200081) | Diagenode',
'meta_keywords' => '5-methylcytosine (5-mC),monoclonal antibody,Methylated DNA Immunoprecipitation',
'meta_description' => '5-methylcytosine (5-mC) Monoclonal Antibody, clone 33D3 validated in MeDIP-seq, MeDIP, DB and IF. Batch-specific data available on the website. Sample size available.',
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'description' => '<p><span>The Auro hMeDIP kit is designed for enrichment of hydroxymethylated DNA from fragmented genomic DNA samples for use in genome-wide methylation analysis. It features</span><span> a highly specific monoclonal antibody against </span><span>5-hydroxymethylcytosine (5-hmC) for the immunoprecipitation of hydroxymethylated DNA</span><span>. It includes control DNA and primers to assess the effiency of the assay. </span><span>Performing hydroxymethylation profiling with the hMeDIP kit is fast, reliable and highly specific.</span></p>',
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<li><span>Robust enrichment & immunoprecipitation of hydroxymethylated DNA</span></li>
<li>Highly specific monoclonal antibody against 5-hmC<span> for reliable, reproducible results</span></li>
<li>Including control DNA and primers to <span>monitor the efficiency of the assay</span>
<ul style="list-style-type: circle;">
<li>5-hmC, 5-mC and unmethylated DNA sequences and primer pairs</li>
<li>Mouse primer pairs against Sfi1 targeting hydroxymethylated gene in mouse</li>
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<li>Improved single-tube, magnetic bead-based protocol</li>
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'meta_description' => 'Auto hMeDIP kit x16 (monoclonal mouse antibody)',
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'description' => '<p><a href="https://www.diagenode.com/files/products/kits/magmedip-kit-manual-C02010020-21.pdf"><img src="https://www.diagenode.com/img/buttons/bt-manual.png" /></a></p>
<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>
<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>
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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>
<div class="small-12 medium-8 large-8 columns">
<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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'info1' => '<p>DNA methylation occurs primarily as 5-methylcytosine (5-mC), and the Diagenode MagMeDIP Kit takes advantage of a specific antibody targeting this 5-mC to immunoprecipitate methylated DNA, which can be thereafter directly analyzed by qPCR or Next-Generation Sequencing (NGS).</p>
<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>
<center><img src="https://www.diagenode.com/img/product/kits/MagMeDIP-workflow.png" width="70%" alt="5-methylcytosine" caption="false" /></center>
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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>
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<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>
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<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>
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<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>
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<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>
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<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>
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<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>
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'meta_title' => 'MagMeDIP Kit for efficient immunoprecipitation of methylated DNA | Diagenode',
'meta_keywords' => '',
'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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'name' => 'Auto MethylCap kit',
'description' => '<p>The Auto MethylCap kit allows to specifically capture DNA fragments containing methylated CpGs. The assay is based on the affinity purification of methylated DNA using methyl-CpG-binding domain (MBD) of human MeCP2 protein. This procedure has been optimized to perform automated immunoprecipitation of chromatin using the <a href="https://www.diagenode.com/en/p/sx-8g-ip-star-compact-automated-system-1-unit">IP-Star® Compact Automated System</a> enabling highly reproducible results and allowing for high throughput.</p>',
'label1' => ' Characteristics',
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<li><strong>Fast & sensitive capture</strong> of methylated DNA</li>
<li><strong>High capture efficiency</strong></li>
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<li><strong>Automation compatibility</strong><strong></strong>
<h3>MBD-seq allows for detection of genomic regions with different CpG density</h3>
<p><img src="https://www.diagenode.com/img/product/kits/mbd_results1.png" alt="MBD-sequencing results have been validated by bisulfite sequencing" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>F</strong><strong>igure 1.</strong><span> </span>Using the MBD approach, two methylated regions were detected in different elution fractions according to their methylated CpG density (A). Low, Medium and High refer to the sequenced DNA from different elution fractions with increasing salt concentration. Methylated patterns of these two different methylated regions were validated by bisulfite conversion assay (B).<br /><strong></strong></p>
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'slug' => 'auto-methylcap-kit-x48-48-rxns',
'meta_title' => 'Auto MethylCap kit x48',
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'meta_description' => 'Auto MethylCap kit x48',
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'id' => '1887',
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'name' => 'MethylCap kit',
'description' => '<p>The MethylCap kit allows to specifically capture DNA fragments containing methylated CpGs. The assay is based on the affinity purification of methylated DNA using methyl-CpG-binding domain (MBD) of human MeCP2 protein. The procedure has been adapted to both manual process or <a href="https://www.diagenode.com/en/p/sx-8g-ip-star-compact-automated-system-1-unit">IP-Star® Compact Automated System</a>. Libraries of captured methylated DNA can be prepared for next-generation sequencing (NGS) by combining MBD technology with the <a href="https://www.diagenode.com/en/p/microplex-lib-prep-kit-v3-48-rxns">MicroPlex Library Preparation Kit v3</a>.</p>',
'label1' => 'Characteristics',
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<li><strong>Fast & sensitive capture</strong> of methylated DNA</li>
<li><strong>High capture efficiency</strong></li>
<li><strong>Differential fractionation</strong> of methylated DNA by CpG density (3 eluted fractions)</li>
<li><strong>On-day protocol</strong></li>
<li><strong>NGS compatibility</strong></li>
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<h3>MBD-seq allows for detection of genomic regions with different CpG density</h3>
<p><img src="https://www.diagenode.com/img/product/kits/mbd_results1.png" alt="MBD-sequencing results have been validated by bisulfite sequencing" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong></strong></p>
<p><strong></strong><strong>F</strong><strong>igure 1.</strong> Using the MBD approach, two methylated regions were detected in different elution fractions according to their methylated CpG density (A). Low, Medium and High refer to the sequenced DNA from different elution fractions with increasing salt concentration. Methylated patterns of these two different methylated regions were validated by bisulfite conversion assay (B).</p>',
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<p style="text-align: center;"><strong>Make your Bisulfite conversion now in only 60 minutes !</strong></p>
<p>Diagenode's Premium Bisulfite Kit rapidly converts DNA through bisulfite treatment. Our conversion reagent is added directly to DNA, requires no intermediate steps, and results in high yields of DNA ready for downstream analysis methods including PCR and Next-Generation Sequencing.</p>',
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<p>Diagenode offers huge selection of highly sensitive antibodies validated in IF.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15200229-IF.jpg" alt="" height="245" width="256" /></p>
<p><sup><strong>Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong></sup></p>
<p><sup>HeLa cells transfected with a Cas9 expression vector (left) or untransfected cells (right) were fixed in methanol at -20°C, permeabilized with acetone at -20°C and blocked with PBS containing 2% BSA. The cells were stained with the Cas9 C-terminal antibody (Cat. No. C15200229) diluted 1:400, followed by incubation with an anti-mouse secondary antibody coupled to AF488. The bottom images show counter-staining of the nuclei with Hoechst 33342.</sup></p>
<h5><sup>Check our selection of antibodies validated in IF.</sup></h5>',
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'description' => '<p><span style="font-weight: 400;">T</span><span style="font-weight: 400;">he pattern of <strong>DNA modifications</strong> is critical for genome stability and the control of gene expression in the cell. Methylation of 5-cytosine (5-mC), one of the best-studied epigenetic marks, is carried out by the <strong>DNA methyltransferases</strong> DNMT3A and B and DNMT1. DNMT3A and DNMT3B are responsible for </span><i><span style="font-weight: 400;">de novo</span></i><span style="font-weight: 400;"> DNA methylation, whereas DNMT1 maintains existing methylation. 5-mC undergoes active demethylation which is performed by the <strong>Ten-Eleven Translocation</strong> (TET) familly of DNA hydroxylases. The latter consists of 3 members TET1, 2 and 3. All 3 members catalyze the conversion of <strong>5-methylcytosine</strong> (5-mC) into <strong>5-hydroxymethylcytosine</strong> (5-hmC), and further into <strong>5-formylcytosine</strong> (5-fC) and <strong>5-carboxycytosine</strong> (5-caC). 5-fC and 5-caC can be converted to unmodified cytosine by <strong>Thymine DNA Glycosylase</strong> (TDG). It is not yet clear if 5-hmC, 5-fC and 5-caC have specific functions or are simply intermediates in the demethylation of 5-mC.</span></p>
<p><span style="font-weight: 400;">DNA methylation is generally considered as a repressive mark and is usually associated with gene silencing. It is essential that the balance between DNA methylation and demethylation is precisely maintained. Dysregulation of DNA methylation may lead to many different human diseases and is often observed in cancer cells.</span></p>
<p><span style="font-weight: 400;">Diagenode offers highly validated antibodies against different proteins involved in DNA modifications as well as against the modified bases allowing the study of all steps and intermediates in the DNA methylation/demethylation pathway:</span></p>
<p><img src="https://www.diagenode.com/img/categories/antibodies/dna-methylation.jpg" height="599" width="816" /></p>
<p><strong>Diagenode exclusively sources the original 5-methylcytosine monoclonal antibody (clone 33D3).</strong></p>
<p>Check out the list below to see all proposed antibodies for DNA modifications.</p>
<p>Diagenode’s highly validated antibodies:</p>
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<li>Highly sensitive and specific</li>
<li>Cost-effective (requires less antibody per reaction)</li>
<li>Batch-specific data is available on the website</li>
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<p>Diagenode has partnered with leading epigenetics experts and numerous epigenetics consortiums to bring to you a validated and comprehensive collection of epigenetic antibodies. As an expert in epigenetics, we are committed to offering highly-specific antibodies validated for ChIP/ChIP-seq and many other applications. All batch-specific validation data is available on our website.<br /><a href="../categories/antibodies">Read about our expertise in antibody production</a>.</p>
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<li><strong>Focused</strong> - Diagenode's selection of antibodies is exclusively dedicated for epigenetic research. <a title="See the full collection." href="../categories/all-antibodies">See the full collection.</a></li>
<li><strong>Strict quality standards</strong> with rigorous QC and validation</li>
<li><strong>Classified</strong> based on level of validation for flexibility of application</li>
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<p>Existing sample sizes are listed below. We will soon expand our collection. Are you looking for a sample size of another antibody? Just <a href="mailto:agnieszka.zelisko@diagenode.com?Subject=Sample%20Size%20Request" target="_top">Contact us</a>.</p>',
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<p><span style="font-weight: 400;">Diagenode’s highly validated antibodies:</span></p>
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<li>Highly sensitive and specific</li>
<li>Cost-effective (requires less antibody per reaction)</li>
<li>Batch-specific data is available on the website</li>
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'name' => 'Exploring the Epigenetic Landscape of Spermatozoa: Impact of Oxidative Stress and Antioxidant Supplementation on DNA Methylation and Hydroxymethylation',
'authors' => 'Elisa Hug et al.',
'description' => '<p><span>Reproductive success is dependent on gamete integrity, and oxidative stress alters male nuclei, meaning that no DNA repair is possible due to chromatin compaction. The composition of sperm makes it highly sensitive to reactive oxygen species (ROS) but, at the same time, ROS are needed for sperm physiology. Over the past 30 years, much attention has been paid to the consequences of oxidative stress on sperm properties and the protective effects of antioxidant formulations to help fertility. Spermatozoa also carry epigenetic marks, critical for embryo development and the health of offspring. As DNA oxidative damage may disturb the sperm epigenome, we used an established mouse model of post-testicular sperm DNA oxidation to investigate sperm DNA methylation and hydroxymethylation. We also analyzed the potential corrective effect of oral antioxidant supplementation, proven to reduce sperm DNA oxidative damage, on sperm DNA methyl/hydroxymethyl marks. We show that sperm DNA oxidation is associated with a significant increase in overall hydroxymethylation. Oral antioxidant supplementation led to unexpected mild epigenetic changes. Antioxidant supplementation should not be proposed without proper clinical evaluation as it may alter sperm epigenetic marks, leading to a risk of paternally inherited epigenetic alterations.</span></p>',
'date' => '2024-12-12',
'pmid' => 'https://www.mdpi.com/2076-3921/13/12/1520',
'doi' => 'https://doi.org/10.3390/antiox13121520',
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'description' => '<p><span>The quest to understand the molecular mechanisms of tumour metastasis and identify pivotal biomarkers for cancer therapy is increasing in importance. Single-omics analyses, constrained by their focus on a single biological layer, cannot fully elucidate the complexities of tumour molecular profiles and can thus overlook crucial molecular targets. In response to this limitation, we developed a multiobjective recommendation system (RJH-Metastasis 1.0) anchored in a multiomics knowledge graph to integrate genome, transcriptome, and proteome data and corroborative literature evidence and then conducted comprehensive analyses of colorectal cancer with liver metastasis (CRCLM). A total of 25 key genes significantly associated with CRCLM were recommended by our system, and </span><i>GNB1</i><span>,<span> </span></span><i>GATAD2A</i><span>,<span> </span></span><i>GBP2</i><span>,<span> </span></span><i>MACROD1</i><span>, and<span> </span></span><i>EIF5B</i><span><span> </span>were further highlighted. Specifically, GNB1 presented fewer mutations but elevated RNA transcription and protein expression in CRCLM patients. The role of GNB1 in promoting the malignant behaviours of colon cancer cells was demonstrated via in vitro and in vivo studies. Aberrant expression of GNB1 could be regulated by METTL1-driven m7G modification. METTL1 knockdown decreased m7G modification in the 3’ UTR of GNB1, increasing its mRNA transcription and translation during liver metastasis. Furthermore, GNB1 induced the formation of an immunosuppressive microenvironment by promoting the CLEC2C-KLRB1 interaction between memory B cells and KLRB1</span><sup>+</sup><span>PD-1</span><sup>+</sup><span>CD8</span><sup>+</sup><span><span> </span>cells. GNB1 expression and the efficacy of PD-1 antibody-based treatment in CRCLM patients were significantly correlated. In summary, our recommendation system can be used for effective exploration of key molecules in colorectal cancer, among which GNB1 was identified as a critical CRCLM promoter and immunotherapy biomarker in colorectal cancer patients.</span></p>',
'date' => '2024-10-24',
'pmid' => 'https://molecular-cancer.biomedcentral.com/articles/10.1186/s12943-024-02155-z',
'doi' => 'https://doi.org/10.1186/s12943-024-02155-z',
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'name' => 'Differential methylation of circulating free DNA assessed through cfMeDiP as a new tool for breast cancer diagnosis and detection of BRCA1/2 mutation',
'authors' => 'Piera Grisolia et al.',
'description' => '<h3 class="c-article__sub-heading" data-test="abstract-sub-heading">Background</h3>
<p>Recent studies have highlighted the importance of the cell-free DNA (cfDNA) methylation profile in detecting breast cancer (BC) and its different subtypes. We investigated whether plasma cfDNA methylation, using cell-free Methylated DNA Immunoprecipitation and High-Throughput Sequencing (cfMeDIP-seq), may be informative in characterizing breast cancer in patients with BRCA1/2 germline mutations for early cancer detection and response to therapy.</p>
<h3 class="c-article__sub-heading" data-test="abstract-sub-heading">Methods</h3>
<p>We enrolled 23 BC patients with germline mutation of BRCA1 and BRCA2 genes, 19 healthy controls without BRCA1/2 mutation, and two healthy individuals who carried BRCA1/2 mutations. Blood samples were collected for all study subjects at the diagnosis, and plasma was isolated by centrifugation. Cell-free DNA was extracted from 1 mL of plasma, and cfMeDIP-seq was performed for each sample. Shallow whole genome sequencing was performed on the immuno-precipitated samples. Then, the differentially methylated 300-bp regions (DMRs) between 25 BRCA germline mutation carriers and 19 non-carriers were identified. DMRs were compared with tumor-specific regions from public datasets to perform an unbiased analysis. Finally, two statistical classifiers were trained based on the GLMnet and random forest model to evaluate if the identified DMRs could discriminate BRCA-positive from healthy samples.</p>
<h3 class="c-article__sub-heading" data-test="abstract-sub-heading">Results</h3>
<p>We identified 7,095 hypermethylated and 212 hypomethylated regions in 25 BRCA germline mutation carriers compared to 19 controls. These regions discriminate tumors from healthy samples with high accuracy and sensitivity. We show that the circulating tumor DNA of BRCA1/2 mutant breast cancers is characterized by the hypomethylation of genes involved in DNA repair and cell cycle. We uncovered the TFs associated with these DRMs and identified that proteins of the Erythroblast Transformation Specific (ETS) family are particularly active in the hypermethylated regions. Finally, we assessed that these regions could discriminate between BRCA positives from healthy samples with an AUC of 0.95, a sensitivity of 88%, and a specificity of 94.74%.</p>
<h3 class="c-article__sub-heading" data-test="abstract-sub-heading">Conclusions</h3>
<p>Our study emphasizes the importance of tumor cell-derived DNA methylation in BC, reporting a different methylation profile between patients carrying mutations in BRCA1, BRCA2, and wild-type controls. Our minimally invasive approach could allow early cancer diagnosis, assessment of minimal residual disease, and monitoring of response to therapy.</p>',
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'pmid' => 'https://translational-medicine.biomedcentral.com/articles/10.1186/s12967-024-05734-2',
'doi' => 'https://doi.org/10.1186/s12967-024-05734-2',
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'description' => '<p><span>Brain metastases (BMs) are the most common and among the deadliest brain tumors. Currently, there are no reliable predictors of BM development from primary cancer, which limits early intervention. Lung adenocarcinoma (LUAD) is the most common BM source and here we obtained 402 tumor and plasma samples from a large cohort of patients with LUAD with or without BM (</span><i>n</i><span> = 346). LUAD DNA methylation signatures were evaluated to build and validate an accurate model predicting BM development from LUAD, which was integrated with clinical factors to provide comprehensive patient-specific BM risk probabilities in a nomogram. Additionally, immune and cell interaction gene sets were differentially methylated at promoters in BM versus paired primary LUAD and had aligning dysregulation in the proteome. Immune cells were differentially abundant in BM versus LUAD. Finally, liquid biomarkers identified from methylated cell-free DNA sequenced in plasma were used to generate and validate accurate classifiers for early BM detection. Overall, LUAD methylomes can be leveraged to predict and noninvasively identify BM, moving toward improved patient outcomes with personalized treatment.</span></p>',
'date' => '2024-10-08',
'pmid' => 'https://www.nature.com/articles/s41591-024-03286-y',
'doi' => 'https://doi.org/10.1038/s41591-024-03286-y',
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'name' => 'RNA m5C oxidation by TET2 regulates chromatin state and leukaemogenesis',
'authors' => 'Zhongyu Zou et al. ',
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