Bisulfite Padlock Probe Sequencing Protocol

Bisulfite padlock probe design, production and sequencing were previously described [16] (link), [43] (link). Briefly, genomic DNA was extracted from peripheral blood of 22 pedigrees, and approximately 1 µg of genomic DNA was bisulfite converted with EZ-96 Zymo DNA Methylation-Gold kit (Zymo Research). Approximately 250 ng of bisulfite converted genomic DNAs were mixed with normalized amount of genome-wide scale padlock probes and oligo suppressors. The padlock probes were annealed to bisulfite converted genomic DNA. The gap between two ends of padlock probes was filled and ligated with AmpliTaq DNA polymerase, Stoffel fragment (Life Technologies) and Ampligase (Epicentre), respectively resulting in circularized DNA. The bisulfite sequencing libraries were generated by library-free BSPP protocol as described [16] (link). Briefly, two-thirds of the circularized DNA of each captured reaction were directly amplified and barcoded with adapter primers compatible with Illumina sequencer. The bisulfite sequencing libraries were purified with AMPure XP magnetic beads (Agencourt), pooled in equimolar ratios, size selected at the size approximately 375 bp with 6% TBE polyacrylamide gel (Life Technologies), and sequenced by Illumina HiSeq2000 and GAIIx sequencers.