GSM2995374: GU-WGBS; Homo sapiens; Bisulfite-Seq
Three micrograms of genomic DNA (extracted from P1-SSP-3, P1-GU-1 and P1-blood) were sheared using Covaris S2 Focused-ultrasonicator, in a 130 µl volume, with 5.0 intensity, 10% duty cycle, 200 cycles per burst, and 6:00 minutes treatment time. Sheared DNA was purified using QIAquick PCR purification kit. Unmethylated DNA of lambda phage was added at 1% concentration to each sample as an internal control of monitoring the conversion efficiency. Libraries were made according to the Illumina library preparation protocol using the methylated adaptors. The libraries were purified using QIAquick PCR purification kit and bisulfite converted using EpiTect bisulfite kit (Qiagen). To increase the conversion efficiency we performed the denaturation steps at 98°C for 10 minutes, and the samples were incubated for another 120 minutes at 60°C at the end of thermocycling reaction. The bisulfite converted libraries were amplified using PfuTurbo HotStart DNA Polymerase (Agilent) for 16 cycles. The libraries were then size selected (200-300bp) and gel purified. The quality of libraries were checked using DNA 1000 Bioanalyzer chip. Libraries were then quantitated by qPCR using KapaBiosystems Kapa Library Quant kit and were run through 101 paired-end Illumina HiSeq 2000.
GEO Accession
GSM2995374