GSM8342380: WGBS_Human_PrimedESCs_Rep2; Homo sapiens; Bisulfite-Seq
Genomic DNA (gDNA) was extracted from hESCs by using DNeasy Blood & Tissue Kits (Qiagen, Cat# 69504) according to the manufacturer's protocol. 200 ng of purified gDNA of hESC was subjected to bisulfite conversion and purification by using the EZ-96 DNA Methylation-Direct MagPrep kit (Zymo Research, Cat# D5045) according to the manufacturer's protocol. Then, WGBS libraries were constructed via our recently published TAILS method. In brief, P5-N6-oligo1 (5'-CTACACGACG-CTCTTCCGATCTN6-3') was used for the first round of random priming in the presence of the Klenow exo(-) fragment. The remaining oligos and dNTPs were removed by Exo-SAP IT Express, and the dC tailing step was performed with the TdT enzyme. Then, a second round of priming was conducted by using P7-G6-oligo2 (5'-AGACGTGTGCTCTTCCGATCTG6HN-3') in the presence of the Klenow exo(-) fragment. After one round of purification with AMPure XP beads, libraries were constructed by PCR amplification. Next, the libraries were purified, and the size distribution was checked on the fragment analyzer. Finally, the WGBS libraries were sequenced on a NovaSeq 6000 sequencer with a 150 bp paired-end sequencing strategy.