title: Whole-genome bisulfite SOLiD 3+ sequencing of normal colonic mucosa 2 of 3
design: Bisulfite sequencing libraries were prepared using the approach previously described by Bormann Chung et al.7, with some modifications so the entire protocol is provided in detail here. 5ug of genomic DNA was sheared using a Covaris E2 sonicator.  40ng of AluI-digested unmethylated ? DNA was added to each sample in order to monitor the efficiency of bisulfite conversion.  Sample DNA ends were then repaired using 1x End Polish Buffer, 400nM each of dATP, dGTP and dTTP (leaving out dCTP), 40 U of End Polishing Enzyme 1 (Life Tech) and 80 U of End Polishing Enzyme 2 (Life Tech).  Adaptor sequences, as detailed by Bormann Chung et al. 7, were then ligated onto the samples, using 1X T4 ligase buffer, 4.5uM of methyl-protect P1 adaptor, 4.5uM of P2 adaptor and 50 U of T4 ligase. Nick translation was performed in 1X Exo-Klenow buffer, 0.5mM dNTPs containing methyl dCTP and 20 units of Exo-Klenow-Fragment (Ambion) for 1 hour at 16 oC. 500ng aliquots of the resulting product was then bisulfite converted using formamide as an additional denaturant8. 24µL of formamide was added to an equal volume of DNA and incubated at 95oC for 5 minutes.  Subsequently, 100µL of Zymo Gold bisulfite conversion reagent (Zymo) was added, and the mixture was incubated for 8 hours in 50oC.  Samples were then desulphonated and purified using spin columns following the EZ-DNA Zymo Methylation-Gold protocol. 5µL of the bisulfite converted library was amplified in 1X PCR buffer, 0.2 mM dNTP, 1mM each of the standard SOLiD fragment library primers, 5 U of Taq (Denville), and 0.25 U of Pfu Turbo Taq (Stratagene). The DNA was subjected to 8 cycles of PCR and the resulting product was purified using AMpure SPRI beads (Beckman Genomics).  The libraries were then sequenced on the SOLiD 3+ platform yielding 50 base pair reads.