Genome-wide DNA Methylation Profiling via RRBS

We used reduced representation bisulfite sequencing (RRBS) to map genome-wide DNA methylation as we have described previously [29 (link),69 (link),70 (link),71 (link)]. Briefly, genomic DNA from each sample (53T, 53C, 60T and 60C) was digested with MspI enzyme followed by end-repair and ligation of Illumina TruSeq sequencing adaptors. Fragments were then size selected (40–220 bp) and bisulfite converted using the EZ DNA methylation direct kit prior to 16–18 rounds of PCR amplification. The quality and size distribution of the RRBS libraries were determined using an Agilent Bioanalyzer and quantified using a Qubit fluorometer. The libraries were sequenced on a single flow cell lane of an Illumina HiSeq 2000 sequencer (100 bp single-ended reads). Base-calling of the reads was performed with Illumina Real Time Analyzer (RTA) software.