GSM5292614: TS108_2_MeDIP_200; Homo sapiens; MeDIP-Seq
Genomic DNA for human methylated DNA immunoprecipitation (MeDIP) was isolated from AML patient blasts using the QIAamp DNA blood Mini Kit (QIAGEN). gDNA samples were diluted in GenDNA TE buffer (Diagenode) to reach a concentration of 100ng/μl and 100μl were sheared in 0.65ml Bioruptor Microtubes (Diagenode). gDNA samples were sonicated for 13 min at high, 30 sec ON, 30 sec OFF settings to 200–300 bp using the Diagenode's Bioruptor platform and the size distribution was confirmed through gel electrophoresis. Barcoded adaptors for Illumina sequencing were added to 1μg of fragmented genomic DNA per experiment, using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs), following the manufacturer's instructions. This protocol was stopped after end repairing, adaptor ligation and clean-up (and before any amplification steps), and the adaptor ligated fragmented DNA was then used for hydroxy-methylated DNA pulldown. This was performed using the MagMeDIP kit (Diagenode) following the manufacturer's instructions. Briefly, magnetic beads were washed twice with ice cold bead wash buffer and incubated overnight with the denaturated adaptor-ligated DNA and the 5-methylcytosine monoclonal antibody 33D3 (C15200081, Diagenode). The day after, DNA IP samples were washed three times in ice-cold MagWash Buffer-1 and once in ice-cold MagWash Buffer-2 and DNA was isolated using the IPure kit v2 (C03010014, Diagenode) following the manufacturer's instructions. Purified methylated genomic DNA was then further processed with NEBNext Ultra II DNA Library Prep Kit for Illumina at the step of PCR enrichment of adaptor-ligated DNA, per the manufacturer's instructions, continuing with the entire protocol to prepare libraries for Illumina sequencing. Before PCR amplification, DNA concentration was calculated using the Qubit dsDNA HS Assay kit (Life Technologies) to determine the number of cycles required for PCR enrichment. Libraries were then barcoded, pooled and sequenced using a NextSeq500 desktop sequencing system (Illumina) with 75- bp, paired-end high output generating 73–90 million reads per sample. Reads were aligned to the human genome (hg38) using BWA-MEM version 0.7.15 (Li and Durbin, 2009).