GSM2722113: medip_9124_d20; Homo sapiens; MeDIP-Seq
DNA was isolated from cell pellets via phenol/chloroform extraction. MeDIP-seq was performed as in Maunakea et al. 2010. Five micrograms of genomic DNA was sonicated to a fragment size of ~100-400 bp using the Bioruptor sonicator (Diagenode). End-repair, addition of 3’-A bases and PE adapter ligation with 2 µg of sonicated DNA was performed according to the Illumina Genomic DNA Sample Prep Kit protocol. Adapter- ligated DNA fragments were size selected to 166-366 bp and purified by gel electrophoresis. DNA was heat denatured and then immunoprecipitated with 5-methylcytidine antibody (Eurogentec; 1 µg of antibody per 1 µg of DNA) in 500 µl of immunoprecipitation buffer (10 µM sodium phosphate, pH 7.0, 140 mM sodium chloride and 0.05% Triton X-100) overnight at 4 °C. Antibody/DNA complexes were isolated by addition of 1 µl of rabbit anti-mouse IgG secondary antibody (2.4 mg ml-1, Jackson Immunoresearch) and 100 µl protein A/G agarose beads (Pierce Biotechnology) for 2 h at 4 °C. Beads were washed nine times with immunoprecipitation buffer and then DNA was eluted in TE buffer with 0.25% SDS and 0.25 mg ml-1 of proteinase K for 2 h at 50 °C. DNA was then purified with the Qiagen QIAquick kit and eluted in 30 µl EB buffer. Ten microliters of DNA was used for a PCR-enrichment reaction with PCR PE Primers 1.0 and 2.0. PCR products were size selected (220-420 bp) and purified by gel electrophoresis. Methylated DNA enrichment was confirmed by PCR on known methylated (SNRPN and MAGEA1 promoters) and unmethylated (a CpG-less sequence on chromosome 15 and glyceraldehyde 3- phosphate dehydrogenase promoter) sequences. DNA libraries were checked for quality by Nanodrop (Thermo Scientific) and Agilent DNA Bioanalyzer (Agilent). Reads were aligned to hg19 using BWA and pre-processed using methylQA (an unpublished C program; available at http://methylqa.sourceforge.net/). Detailed library construction protocols for MeDIP-seq are publicly available at the NIH Roadmap Epigenomics project website (http://www.roadmapepigenomics.org/protocols/type/experimental/).