Instrument: Illumina NovaSeq 6000
Strategy: MeDIP-Seq
Source: GENOMIC
Selection: 5-methylcytidine antibody
Layout: PAIRED
Construction protocol: DNA was extracted and RNAse treated using DNeasy Blood and Tissue kit. To fragment DNA for immunoprecipitation, 8µg of DNA per sample was digested using MseI and DraI restriction. Resulting fragments were end repaired using the large (Klenow) fragment of DNA polymerase. Purified, fragmented DNA (3 µg) was diluted in 450 µL of ultra-pure water, and heat denatured at 95°C for 10 minutes. Samples were then immediately placed on ice to prevent re-annealing of fragments. Following cooling, 50 µL of 10x immunoprecipitation (IP) buffer and 4µL of 5-methyl cytosine (5meC) antibody. Samples were then incubated overnight with rotation at 4°C. For each sample, 50 µL of MagnaBind Goat Anti-Mouse IgG magnetic beads were added to each sample and incubated with rotation for 2 h at room temperature. Following incubation, beads were collected using a magnetic rack, and supernatent discarded. Beads were then washed three times in 1X IP buffer, and resuspended in in 500μL of proteinase K digestion buffer containing 5μL/mL proteinase K solution (stock concentration 20 mg/mL). Proteinase K reactions were incubated with rotation for 2 h at 50°C. After incubation, beads were collected using a magnetic rack and the supernatant collected. To purify immunoprecipitated DNA, the supernatant from each reaction was PCI purified according to published protocols. Libraries were prepared using ACCEL-NGS® 1S Plus & Methyl-SEQ kit (Swift Biosciences, Ann Arbor, MI.) and sequenced on an Illumina NovaSeq to generate 2 x 150 bp reads.