Bisulfite sequencing (after MethylCap) of the SW403 colon carcinoma cell line aligned against HG19 using BS Seeker.
DOWNLOAD (Right click and save link as.)
Experimental protocol:
Methylated DNA was captured by MethylCap kit (Diagenode) as per manufacturer’s instruction. Briefly, 2ug of genomic DNA was randomly sheared by sonication using Bioruptor (UCD-200, Diagenode) to be peaked around 300bp. DNA was incubated with H6-GST-MBD (Methyl-DNA Binding Domain) rotating at 4C for 2 hours before adding capture magnetic beads. After incubating another one hour, capture beads were subjected to serial washing before the captured methylated DNA was eluted low-salt, medium-salt and high-salt buffers in the kit. Medium salt and high salt elute was combined for each sample for the subsequent Bisulfite Sequencing. To construct DNA library for bisulfite sequencing, we used Illumina ChIP seq sample preparation kit with modification. Briefly, methylation enriched DNA were treated as per manufacturer’s instruction before ligating the methylated adaptor (Illumina) instead of regular adaptor. DNA was then bisulfite treated using EZ DNA methylation-gold kit (cat#: D5006, Zymo Research) before amplified using Illumina PE 1.0 and PE 2.0 primers for 14 cycles. Constructed libraries were quantitated using Illumina library quantitation qPCR protocol and sequenced on GAIIx (Illumina) at 36bp.
Then, reads were aligned against the reference genome HG19 using BS Seeker allowing 2 mismatches.