Whole-Genome Bisulfite Sequencing
5ug of gDNA was sheared to 700bp using the focused-ultrasonicator S2 (Covaris) and spiked in with 50ng HaeIII digested lambda DNA for quality control. The fragments were treated with end-repair, A- tailing, and ligation of methylated Illumina adaptors (Illumina) using the DNA library preparation kit for Illumina (Kapa Biosystems). The adaptor ligated DNA was subjected to bisulfite treatment using EpiTect Bisulfite Kit (Qiagen). The bisulfite-treated DNA was amplified with 8 cycles of PCR with uracil-insensitive Taq polymerase (Pfu Turbo Cx Hotstart DNA polymerase; Stratagene). The PCR product was run on a 2% agarose gel followed by gel excision to select for fragments between 300 to 400bp. Purified library was sequenced on the Illumina Hiseq. From 118,292,962 paired reads generated, 109,693,334 paired reads (92.73%) survived adaptor trimming. Out of these reads, 83% was aligned to the genome by BSMAP and 71% properly paired. Assuming all cytosine at non-CpG are not methylated and will be converted, the estimated conversion rate is ~ 98%. 73% of the genome is covered by ≥ 5 non-redundant reads.