Reduced Representation Bisulfite Sequencing

Reduced Representation Bisulfite Sequencing (RRBS) was performed as described [7 (link)]. Briefly, DNA samples were incubated with MspI enzyme (NEB) in order to fragment DNA at CCGG positions to enrich for CpG regions. Bisulfite conversion was performed using the EZ DNA methylation Kit (Zymo Research) according to the manufacturer’s instructions. DNA was then PCR amplified and ligated to TruSeq (Illumina) sequencing adapters. Libraries were subjected to 75-bp single-end sequencing on a NextSeq 550 (Illumina). On average, 18.3 million reads/sample were aligned and 205304 CpGs had sufficient coverage in every sample.