GSM8657417: HeLa_RNASEH; Homo sapiens; OTHER
The genomic DNA was purified from HEK293T or mES cells via phenol/chloroform extraction and ethanol precipitation, or genomic DNA extraction kit. 1 µg DNA was incubated with NEB restriction enzyme mix (Alu I, Mbo I, Mse I, Dde I), Nuclease P1, T5 exonuclease, Lambda exonuclease at 4 °C for 2 hr. As controls, RNase H (NEB, M0297S) was added during the incubation stage. 0.2 µL of Proteinase (Thermo Scientific, EO0491) was added before incubation at 55 °C for 30 min and 70 °C for 30 min. 0.3 µL 1M Tris-HCl (pH 8.0), 0.15 µL 1M MgCl2, 1.5 µL PEG200, 4.8 µL 50% PEG8000 and 1 µL transposase (Vazyme, TD501) were added and incubated at 55 °C for 10 min. The transposition was stopped by the addition of 5 µL 0.25% SDS and incubation for 5 min at room temperature. Transposed DNA was gap-filled by addition of 2 µL Bst3.0 DNA polymerase (NEB, M0374S) and 25 µL NEBNext Q5 HotStart HiFi PCR Master Mix (NEB, M0543S). The mixture was incubated at 72 °C for 15 min and 80 °C for 10 min. For library amplification, the mixture was mixed with a barcoded i5 primer and a barcoded i7 primer PCR was performed to amplify the libraries using the following PCR conditions: 98 °C for 45 s; thermocycling for 10 cycles at 98 °C for 15 s, 65 °C for 75 s; followed by 65 °C for 5 min. The final libraries were purified with the 1× AMPure beads (Beckman, A63882) size selection and were subjected to next-generation sequencing.