claps-seq of human:U2OS WT
U2OS cells cultured to 70%80% confluency in one 150-mm plate per sample and treated with 20 mM of KBrO3 (Sigma-Aldrich, 60085) and 2.5 mM of glutathione (Sigma-Aldrich, V900456). After incubation for 30 min, medium was removed and cells were washed three times with DPBS (Sigma-Aldrich, D8662). Then cells cultured in fresh medium for 1 h, 3 h and 12 h before harvested by centrifugation. Genomic DNA was extracted by DNAzol for CLAPS-seq. For in vitro oxidation (oxidized naked DNA), DNA from untreated cells was incubated with 1 mM of KBrO3 and 2 mM of glutathione in sodium phosphate buffer (10 mM, pH 7.8) at 37 for 80 min. Fresh DNA was labeled with potassium hexabromoiridate (K2IrBr6, ThermoFisher Scientific, 012651) and amine-tagged biotin probe (Amine-(PEG)11-biotin, ThermoFisher Scientific, 26136). Purified biotinylated DNA was sonicated to generate fragments averagely 600 bp in length. DNA fragments (up to 5 g) were ligated to adapter 1 (Ad1) by NEBNext Ultra II DNA Library Prep Kit (New England Biolabs, E6040) and attached to Dynabeads MyOne Streptavidin C1 beads. After thorough wash, extension primer O3P was attached to ligated fragments which were still bound on beads and extended by KAPA HiFi DNA polymerase, followed by ExoI treatment and ended by equal volume of 1% SDS. Next, the beads were thoroughly washed, followed by elution with fresh NaOH to release the complementary strands and neutralized by acetic acid. Purified extension products were ligated to adapter 2 (Ad2), followed by PCR amplification. Libraries were sequenced in PE150 format on Illumina NovaSeq platform by Mingma Technologies Company.