GSM1134680: Brain; Homo sapiens; Bisulfite-Seq
DNA extracted by Phenol:Chloroform:Isoamylalcohol (Sigma) BS-seq library constructing, 10ug genomic DNA was fragmented using a Covaris sonication system (Covaris S2). Following fragmentation, libraries were constructed using the Illumina Paired-End protocol consisting of end repair, <A> base addition and methylated-adaptor ligation. Ligated DNA was bisulfite converted using the EZ DNA Methylation-Gold kit (ZYMO). Different insert size were excised from the same lane of a 2% TAE agarose gel. Normally, three bands are excised corresponding to DNA insert sizes of 80-100 bp, 100-120 bp to 120-150bp. Products were purified by using QIAquick Gel Extraction kit (Qiagen) and amplified by PCR. PCR was carried out in a final reaction volume of 50ul consisting of 20ul purified DNA, 4ul 2.5 mM dNTP, 5 ul 10X buffer, 0.5 ul JumpStart Taq DNA polymerase, 2ul 10uM PCR primers and 37.5 ul ÎœltraPure TM Water and the following thermal cycling program: 94C 30 s, 10 cycles of 94C 30 s, 60C 30 s, 72C 30 s then prolong with 1 min at 72C. PCR products were sequenced with an Illumina genome analyzer.