GSM4907398: DKO1_Input (G4-seq); Mus musculus; OTHER
Briefly, ~10 million cells were fixed with 1% formaldehyde at room temperature with nutation for 10 minutes mins at 1x106 cell/mL in media, quenched with 125 mM glycine, washed twice with ice cold PBS. Cells were pelleted, snap-frozen with liquid nitrogen, and stored at -80°C until use. Cell pellets were then lyzed using the Chromatrap ChIP hypotonic solution (800 ul) and lysis buffer (150ul) supplemented with protease inhibitor cocktail (invitrogen) on ice according to manufacturer's instructions. The nuclei suspension was then sonicated using the bioruptor pico sonicator (Diagenode), 16 cycles 30 seconds on / 30seconds off and the lysates were then cleared by centrifugation at >12000g for 10 minutes. The lysates were diluted 1:10 times in intracellular buffer (25 mM HEPES pH 7.5, 10.5 mM NaCl, 110 mM KCl, 130 nM CaCl2, 1 mM MgCl2) (total 200 ul) then treated with 1mg/ml RNASE A (AM2269; Ambion, 1:100 dilution) for 20 minutes at 37C. Chromatin lysates were then incubated with BG4-Ig antibody (Ab00174-1.1, 1mg/ml; Absolute Antibody) (1:100) for 1.5 hours at 16 C followed by incubation with protein G magnetic dynabeads (Invitrogen). The beads were then washed 5 times with 1 ml wash buffer (intracellular buffer with 0.05% tween 20). The beads were re-suspended in elution buffer (100 mM NaHCO3, 1% SDS, 1 mg/mL RNaseA; Qiagen) treated with RNASE A (1:100, 100mg/ml) at 37 C for 30 minutes and proteinase K (1:40, 20mg/ml; Ambion) at 65 C for 2 hours to overnight. DNA was purified with Zymo ChIP DNA Clean & Concentrator-Capped Column (Zymo Research, Irvine, CA). Library was prepared with NEB Ultra II library prep kit (NEB) following manufacturer's instruction and was sequenced on an Illumina NovaSeq (50 bp paired-end reads).