title: GSM8073541: NUP98_PHF23_WT,MLL1,ChIP-seq; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were fixed in 1% formaldehyde (Thermo Scientific, 28908) for 10min, followed by quenching with 125mM glycine for 5min. Cells were then washed twice with cold PBS added with protease inhibitors (Sigma-Aldrich, 4693132001), and then subjected to resuspension and incubation in LB1 buffer (50 mM HEPES-KOH pH 7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100). The cell nuclei were collected for sonication using Bioruptor sonicator (Diagenode, B01020001; at high-energy setting for 45 cycles with 30s on and 30s off). Supernatant was collected after centrifugation (20,000g for 10min at 4 °C) for incubation with the dynabeads (Invitrogen, 11204D) that are pre-bound with antibodies for 8h at 4 °C. After a series of wash with LB1 buffer, LB2 buffer (10 mM Tris-HCl pH 8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA), and LB3 buffer (10 mM Tris-HCl pH 8.0, 100mM NaCl, 1 mM EDTA, 0.5mM  EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine), the chromatin–protein complexes bound to beads were eluted, subject to reverse crosslink overnight at 65 °C, and treated with RNase (Roche, 11119915001; 1 h at 37 °C) and then protease K (Roche, 03115828001; 2h at 55 °C). The final DNA sample, as well as 1% of input chromatin, was recovered using PCR purification kit (Qiagen, 28106). The ChIP–seq library was prepared using NEBNext Ultra II kit (NEB, E7645L) following the manufacturer's instructions.