EZ DNA Methylation kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
For best results, the CT Conversion Reagent should be prepared freshly and used immediately following preparation.	Always include fully methylated (in-vitro methylated DNA) and non-methylated controls (Leucocyte DNA, DNMT1/3 double KO DNA) during MSP

Upstream tips
For best results, the CT Conversion Reagent should be prepared freshly and used immediately following preparation.
Protocol tips
Always include fully methylated (in-vitro methylated DNA) and non-methylated controls (Leucocyte DNA, DNMT1/3 double KO DNA) during MSP

Publication protocol

Methyl-DNA immunoprecipitation sequencing (MeDIP-Seq)
Before carrying out MeDIP, we sonicated genomic DNA to produce random fragments ranging in size from 100 to 500 bp and purified using the PCR purification kit (Qiagen). Based on the manufacturer's recommended protocol, we then end-repaired, phosphorylated and A-tailed the fragmented DNA and ligated Illumina single read adapters to the fragments. We used ∼4 µg of adaptor-ligated DNA for subsequent MeDIP enrichment. Briefly, following adaptor ligation, DNA was denatured at 95°C for 10 min. Immunoprecipitation was then carried out at 4°C for 3 hr using 10 µg of monoclonal antibody against 5-methylcytidine (Eurogentec) in a final volume of 500 µl IP buffer (10 mM sodium phosphate (pH 7.0), 140 mM NaCl, 0.05% Triton X-100). We incubated the mixture with 40 µl of Dynabeads with M-280 sheep antibody to mouse IgG (Dynal Biotech) for 2 hr at 4°C and washed it seven times with 700 µl of IP buffer. We then treated the beads with proteinase K for 4 hr at 50°C and recovered the methylated DNA by phenol-chloroform extraction followed by ethanol precipitation. PCR amplification by Illumina single read PCR primers was performed as described earlier. We performed agarose gel electrophoresis and excised bands from the gel to produce libraries with insert sizes of ∼200 bp, and quantified these libraries using the Quant-iT PicoGreen dsDNA Reagent and Kits (Invitrogen). We then prepared flowcells with 14 pM DNA using the manufacture's recommended protocol and sequenced for 36 cycles on an Illumina Genome Analyzer II.

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Papers

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Manufacturer protocol

Download the product protocol from Zymo Research for EZ DNA Methylation kit below.

Download manufacturer protocol

Videos

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