5hmC-Seal was performed on genomic DNA isolated from WT H1 hESCs. Briefly, 100 ng gDNA was fragmented, followed by A-tailing and ligation with sequencing adapters using the KAPA Hyper Prep Kit (KAPA Biosciences). Next, gDNA 5-hmC residues on fragments were subjected to T4 β-glucosyltransferase (Thermo) enzymatic modification with UDP-N3-glucose (Active Motif), followed by subsequent chemical modification with biotin-PEG4-dibenzocyclooctyne (DBCO, Sigma). Biotinylated gDNA fragments were separated using streptavidin M270 Dynabeads (Invitrogen) and PCR amplified. Constructed sequencing libraries were purified with AMPure XP beads (Beckman Coulter), and paired-end sequencing was performed using the Illumina Novaseq 6000 sequencer.
GEO Accession
GSM4520692