SuBLiME enriched bisulfite-treated SW-480
DNA was first sheared to a size of about 100-300 bp and ligated with modified adaptor P2 linkers. The standard SOLiD adaptor P2 sequence contains only four cytosines in strand A. These were replaced with other nucleotides (P2-BtnA: 5'-GAGAATGAGGAATGTGGGGTAGGTT and P2-BtnB: 5'-CCTACCCCACATTCCTCATTCTCT) so that the modified P2 adaptor no longer contained any cytosines on the A strand. This is important for subsequent specific labelling in the presence of biotin dCTP. The linkered DNA was then cut with the restriction enzyme Csp61 (5’-C’TAG) and ligated with a modified adaptor P1 (P1-BtnAM: 5'-MMAMTAMGMMTMMGMTTTMMTMTMTATGGGMAGTMGGTGAT, P1-BtnB: 5'-TAATCACCGACTGCCCATAGAGAGGAAAGCGGAGGCGTAGTGGCC). Here, in strand A of the adaptor (P1-BtnAM), cytosines were replaced by 5 methylcytosines (denoted by 'M') so that they would resist modification during bisulfite treatment. This results in formation of a directional library in which one end of each fragment (adaptor P2) is random and the end from which sequencing is initiated (adaptor P1) is formed at a Csp61 site. The linkered DNA fragments were then denatured and reacted with sodium bisulfite under standard conditions. Strand B of adaptor P2 (P2-BtnB) was used to prime synthesis of the strand complementary to the bisulfite-treated DNA and the original DNA strand then degraded using uracil deglycosylase. Following this, adaptor 1 strand A (P1-BtnA) was used to prime DNA synthesis in the presence of biotin dCTP. Since the adaptor P2 sequence has been modified to remove cytosines from this strand, the only incorporation of biotin occurs at positions corresponding to methylated cytosines. DNA synthesis steps were done using Taq polymerase with extension at 65°C for reasons that strand-displacement synthesis would result in incorporation of biotin dCTP into the complementary guanine-rich strand.  After capture of the biotin-containing DNA using streptavidin magnetic beads, DNA was eluted and amplified for a minimal number of cycles using standard SOLiD linkers prior to 50 base deep sequencing using SOLiD-3 chemistry.