Hairpin Bisulfite-Seq Library Construction

Hairpin bisulfite-seq library construction was performed according to previously described protocol21 (link) with slight modifications. Briefly, 10 ug genomic DNA of each sample was spiked with 0.02% unmethylated Lambda DNA (Promega) and sonicated to 200 bp fragments with Covaris. After MseI and MluCI digestion (NEB), end repair and dA tailing, genomic DNA fragments were ligated to Biotin-modified hairpin adapter (5′P-GGCCAGCTGCA AG/iBiodT/GAAGCAGCTGGCCT-3′, IDT). After captured with Dynabeads^® MyOne™ Streptavidin C1 beads (Invitrogen), genomic DNA fragments were subjected to bisulfite conversion using the EpiTect Bisulphite Kit (Qiagen), PCR, and pair-end sequenced using Illumina MiSeq and HiSeq 2000. Illumina Sequencing services were performed at the genomic core of Virginia Bioinformatics Institute.