title: GSM8991512: ASXL1_TR_OE_input; Homo sapiens; ChIP-Seq
library_construction_protocol: ChIP-seq: Cells were washed with PBS and crosslinked in 1% formaldehyde (Thermo Scientific #28908) for 10 minutes, then quenched with 125 mM glycine and 0.5% BSA for 5 minutes. Cells were washed twice with cold 0.5%BSA/PBS and resuspended in Resuspension buffer (9 mM HEPES-KOH pH7.9, 76.4 mM KCl, 0.9 mM EDTA, 0.5% Igepal CA-630). The isolated nuclei were pelleted and resuspended in Lysis Buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.4% Sodium deoxycholate, 0.1% SDS, 1% Igepal CA-630, 0.5 mM DTT, 1 mM PMSF and 1  protease inhibitor cocktail), then sonicated for 12 cycles (30 seconds on/30 seconds off) using a Bioruptor (Diagenode).The lysates were clarified by centrifugation (20,000g, 10 minutes, 4 °C), and the supernatants were incubated with the magnetic beads: Pierce™ Anti-HA Magnetic Beads (Invitrogen #88836) for ASXL1 or Protein A Mag Sepharose (Cytiva #28951378) with BRD2 antibody (Abcam ab13960). Following a series of washes, chromatin–protein complexes were eluted and reverse-crosslinked, followed by RNase A and protease K digestion. DNA was purified using AMPure XP (Beckman, A63881), alongside 1% input control. ChIP-seq libraries were prepared using the NEBNext Ultra II kit (NEB #E7645S) following the manufacturer's instructions.CUT&Tag was performed in ASXL1-HA U2OS knock-in cells using the NovoNGS® CUT&Tag 4.0 High-Sensitivity Kit (N259-YH01) according to the manufacturer's protocol.ATAC-seq in U2OS knock-in cells was performed using High-Sensitivity Open Chromatin Profile Kit 2.0 (N248), according to the manufacturer's instructions.