GSM9549362: MCM10 ChIP-seq, +RECQL4, Rep2 (ChIP); Homo sapiens; ChIP-Seq
ChIP-seq analysis was performed according to previously established methods (Zhu et al. 2025). Approximately 2 × 107 cells were trypsinised, washed twice with 10 ml PBS, and resuspended in 1 ml PBS for crosslinking. This was achieved by adding an equal volume of fresh 2% paraformaldehyde (10 min, room temperature), followed by quenching with 125 mM glycine for 5 min. After sequential washes with PBS, PBS/0.5% NP-40, and PBS/10% glycerol, cell pellets were either flash-frozen in liquid nitrogen for storage at -80 °C or lysed immediately. For lysis, cells were incubated on ice for 10 min in 1 ml ChIP lysis buffer (25 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.5% Sarkosyl, 0.1% sodium deoxycholate, 0.5% Triton X-100, and Roche protease inhibitors). Chromatin fragmentation was performed using a Covaris S2 focused-ultrasonicator (5% duty factor, 100 cycles/burst, intensity 3) for 10 min. Following centrifugation (18,400 x g, 15 min, 4 °C), the supernatant was pre-cleared with 1.5 µg Protein G Dynabeads (Invitrogen, #10004D) for 1–2 h. A 2% input aliquot (20 µl) was reserved, while the remaining supernatant underwent immunoprecipitation overnight at 4 °C with 5 µg anti-FLAG M2 antibody (Sigma, #F1804) bound to Protein G Dynabeads. The beads were washed stringently with RIPA buffer, RIPA containing 300 mM NaCl, RIPA with 250 mM LiCl, and twice with TE buffer. DNA was eluted twice at 65 °C in TE + 1% SDS, and crosslinks were reversed at 65 °C for 4 h following RNase A and Proteinase K treatment. Finally, DNA was purified using the Takara NucleoSpin Gel and PCR Clean-up Kit. Libraries were generated using the NEBNext Ultra II kit for 50 bp paired-end sequencing on an Illumina NovaSeq 6000.