Low coverage bisulfite sequencing of human colon normal tissue
Total genomic DNA was isolated using the DNAeasy Blood & Tissue Kit (Qiagen) and sheared using the Covaris LE220. Ampure XP beads (Agencourt) were then used to size select gDNA fragments within 150-320bp, and sheared distribution was verified via BioAnalyzer (Agilient) prior to continuation. Sheared genomic DNA (1 ug) was then generated into a sequencing library via end repair, 3' A base tailing (KAPA Hyper Prep Kit #KK8502) and sequencing adapter ligation (Roche SeqCap Epi Enrichment System). Post-ligation clean-up was performed using Ampure XP beads. Following bead clean-up, DNA library was then bisulfite converted using the EZ DNA Methylation-Lightning Kit (Zymo Research) and amplified via PCR using KAPA HiFi U+ HotStart ReadyMix (KAPA #KK2800). Equal concentration of each bisulfite-converted library was then combined in sets of either three or four libraries/per pool along with SeqCap Epi universal & indexing oligos and bisulfite capture enhancer (SeqCap Epi Accessory Kit). Each pool was subsequently lyophilized using TOMY Micro-Vac (MV100) and resuspended in hybridization buffer (SeqCap Epi Hybridization and Wash Kit) prior to being hybridized to SeqCap Epi Probe Pool (Roche) for 72 hours at 47C in a thermocycler with a heated lid at 57C. Following the 72-hour incubation, captured bisulfite-converted libraries were recovered (SeqCap Pure Capture Bead Kit) at 47C in a thermocycler for 45 minutes, with intermediate vortexing every 15 minutes. Capture beads were then washed (SeqCap Hybridization and Wash Kit) in a 47C water bath at room temperature, respectively. Captured bisulfite-converted libraries were amplified via PCR (SeqCap Epi Accessory Kit). Libraries were sequenced with 10% PhiX spike-in as 100-base paired end reads on the HiSeq2500 in rapid run mode.