For 5hmC profiling genomic DNA was isolated from HUES8 WT and HUES8 TKO hESCs using the DNeasy Blood & Tissue Kit (Qiagen, 69504) following manufacturer’s guidelines. 40 μg of genomic DNA was sonicated to ~200-400bp fragments using a Diagenode Bioruptor Sonicator. Sonicated DNA was then labeled with azide glucose in a 1 hr reaction at 37°C catalyzed by recombinant -GT utilizing UDP-6-N3-glucose as the sugar donor. The reactions were cleaned up using a Zymo DNA Clean & Concentrator kit (Zymo, D4003), then a biotin moiety was added to the azide-labeled DNA via a copper-free click chemistry reaction with DBCO-S-S-PEG3-Biotin in water at 37°C for 1 hr. Reactions were once again cleaned with the Zymo kit and then bound to Dynabeads MyOne Streptavidin C1 beads (Life Technologies, 65001) for 15 minutes at RT and washed 5 times with binding buffer (5mM Tris-HCl, pH7.5, 0.5mM EDTA, 1M NaCl, 0.01% Tween 20). Bound DNA was eluted by reducing the disulfide in the biotin linker with 100mM DTT for 2h at RT with gentle rotation. Eluted DNA was cleaned on a Micro Bio-spin Column (BioRad, 7326204) to remove DNA and then purified by the Zymo kit. Libraries were constructed from eluted DNA by end repair, A-tailing, and adapter ligation, followed by 4 cycles of PCR, size selection via agarose gel electrophoresis, 12 additional PCR cycles, and a final size selection via agarose gel electrophoresis. Library quantity and quality were analyzed by Bioanalyzer prior to sequencing.