title: Illumina GAII capture bisulfite sequencing (using padlock probes) of normal colonic mucosa 3 of 3
design: To obtain accurate quantitation of absolute DNA methylation level at the single-nucleotide resolution, we used the Bisulfite Padlock Probes (BSPP) previously developed. In this method, a library of long oligonucleotide sequences is designed for the regions of interest, synthesized in situ using Agilent’s Inkjet printing technology, and enzymatically converted into single-strand padlock probes.  A BSPP library was custom designed based in part on DMRs previously found in colon cancer, covering ~60,000 highly curated differentially methylated regions in the human genome (620,708 CpG sites in 19.2Mb of genomic regions covered).   1µg of genomic DNA from the three tumor-normal samples was bisulfite converted using the Zymo EZ Methylation Gold Kit.  The bisulfite converted DNA was then captured with BSPP using a previously described method.   Briefly, 300ng of bisulfite converted DNA was captured in a 10µl reaction containing 1X Ampligase Buffer (Epicenter Biotechnologies).  The mixture was heated to 95°C for 30 seconds and then incubated for 20 hours at 58°C.   A 2µl mix containing 2U/µl AmpliTaq Stoffel fragment, 0.5U/µl Ampligase, 50µM dNTP was added to each tube.  The tubes were incubated at 58°C for another 28 hours and then heat inactivated at 94°C for 2 minutes.  Single stranded DNA was removed by adding 2µl of Exo I/III mix and incubating at 37°C for 1 hour.  300ng of the captured padlock probes were amplified as previously described and then digested with Mme I in order to remove the amplification primers9.  Adapters compatible with the Illumina GAII sequencer were then ligated to the digested DNA in order to generate the sequencing libraries.