GSM7068447
HEK293T and HT1080 cells were cultured in the presence of 100 μM BrdU (sigma) for 2 h. To obtain four sub-populations of S phase cells, including S1, S2, S3 and S4, cells were fixed, stained with PI and sorted by FACS according to their DNA content. Next, isolated cells were lysed immediately in lysis buffer (50 mM Tris, pH 8.0, 10 mM EDTA, 1 M NaCl, 0.5% SDS, 0.1 mg/mL RNase A, 0.2 mg/mL Protease K) and incubated at 37 °C for 3 h, then at 55 °C for 8 h. Genomic DNA was extracted with phenol: chloroform: isoamyl (25:24:1) and chloroform: Isoamyl alcohol (24:1) and precipitated with 75% ethanol and 300 mM NaAc. DNA pellet was dissolved in TE (10 mM Tris, pH 8.0, 1 mM EDTA) before being sonicated using Bioruptor to 200-500 bp. The sonicated DNA was end-repaired, dA-tailed, and adapter-ligated. The adapter-ligated DNA was boiled for 10 min and cooled on ice immediately for 2 min. DNA solution was incubated with BrdU antibody (BD Bioscience, BD44) and rotated at 4 °C overnight. The mixture was incubated with protein G Dynabeads (Life Technology, 10009D) and rotated at 4 °C for another 8 h. After washing five times with washing buffer (PBS with 0.05% (v/v) Triton-X100) and once with TE, the beads were eluted by elution buffer (TE with 0.5% (w/v) SDS) at 65 °C for 10 min. After extraction with DNA Clean & Concentrator-5 kit (ZYMO, D4014), DNA was subjected to library amplification using Next Q5 Hot Start HiFi PCR Master Mix (NEB, M0543) and NEBNext Multiplex Oligos for Illumina (NEB, E7600). The libraries were purified with Ampure beads (0.9 ×) (Beckman, A633881) and sequenced using DNBSEQ-T7 platforms.