5hmC Chemical Labeling and Enrichment

The 5hmC-Seal was performed as previously described in Han et al.^{30 (link)}. Briefly, 100 ng genomic DNA were fragmented in Tagmentation buffer at 55 °C. Fragmented DNA was purified by Zymo DNA Clean and Concentration Kit. Then, the selective 5hmC chemical labeling was performed in glucosylation buffer (50 mM HEPES buffer pH 8.0, 25 mM MgCl₂) containing above fragmented DNA, βGT, N3-UDP-Glc, and incubated at 37 °C for 2 h. After DNA purification in ddH₂O, DBCO-PEG4-Biotin (Click Chemistry Tools) was added and incubated at 37 °C for 2 h. The biotin-labeled DNA was pulled down by C1 Streptavidin beads (Life Technologies) for 15 min at room temperature. Next, the captured DNA fragments were subjected to PCR amplification using Nextera DNA sample preparation kit. The resulting amplified product was purified by 1.0X AMPure XP beads. Input library was made by direct PCR from fragmented DNA directly without labeling and pull-down. The libraries were quantified by a Qubit fluorometer (Life Technologies) and sequenced on NextSeq 500.