GSM8342375: ACE_Human_PrimedESCs; Homo sapiens; OTHER
Genomic DNA (gDNA) was extracted from human embryonic stem cells by using DNeasy Blood & Tissue Kits (Qiagen, Cat# 69504) according to the manufacturer's protocol. 200 ng of gDNA was then subjected to the optimized ACE-seq protocol. The genomic DNA was first fragmented and then subjected to the ACE-seq library construction protocol with modifications. Briefly, fragmented gDNA was glycosylated by using UDP-glucose and T4 Phage β-glucosyltransferase (New England Biolabs, Cat# M0357) at 37°C for 1 hr. Afterwards, 0.1 M NaOH was added for denaturation at 55°C for 10 mins. The APOBEC enzyme (New England Biolabs, Cat# E7125) was used to deaminate gDNA at 37°C for 3 hrs. After deamination, gDNA was purified once using AMPure XP beads (Beckman coulter, Cat# A63882) before library construction following to the TAILS procedure. Fragment Analyzer (Agilent 5200) was used to check the size distribution of the final libraries. Finally, the ACE-seq libraries were sequenced on a NovaSeq 6000 sequencer by a 150 bp paired-end sequencing strategy. Additionally, in vitro CpG-methylated lambda DNA (Thermo Fisher Scientific, Cat# SD0021) was used as a spike-in to estimate the deamination rate in the 5mCpG context and unmodified CH context (H=A, C, T). In addition, we incorporated fully hydroxymethylated DNA to assess the protection rate of 5hmC by glycosylation in individual samples.