GSM5580231: HEK293T cells, Input_rep1; Homo sapiens; OTHER
Cells were harvested and genomic DNA was extracted by Monarch® Genomic DNA Purification Kit (NEB, T3010S). Butylated hydroxytoluene (0.1 mM) (sigma) was added to prevent further DNA oxidation during DNA extraction. Genomic DNA was digested to 100–300 bp with NEBNext dsDNA Fragmentase (NEB, M0348) following the manufacturer's guidelines. The genomic DNA was labelled with  NTHL1 in  Tris-HCl buffer (50 mM, pH 7.0) with 1 mM EDTA, 1mM DTT, and 10 mM NaBH3CN at 37°C for 60 min. Mixtures of glycosylase and genomic DNA were enriched by incubating with 20 μL HisPur Ni-NTA Resin in 800 μL PBS with 20 μg single-stranded salmon sperm DNA. Then the mixture was placed at 4°C overnight. After centrifugation at 8000 rpm, the supernatant was discarded, the beads were washed twice with 1 mL PBS and then twice with 1 mL washing buffer (20 mM Tris-HCl pH 7.5，0.2% tween). NEB adaptor was ligated to double-stranded DNA on Ni-NTA resin to remove the free adaptor by beads washing. The ligation assay was performed by following the manufacturer's guidelines. After completing the adaptor ligation, beads were washed twice with PBS and twice with washing buffer. After washing, DNA was eluted from the beads using 20 μL PBS with 500 mM imidazole at 37°C for 30 min with interval mixing at 1,200 rpm. The subsequent enrichment procedures (SDS-PAGE and membrane transfer) were the same as procedures described above. Purified DNA was subjected to qPCR and bioanalyzer analysis for quantification and verification. The library was then constructed using NEBNext Ultra DNA Library Prep Kit (NEB, E7103S) following the manufacturer's instructions.