GSM7886624: ex vivo expanded splenic Ikzf1 KO NK cells, Ikzf1 ChIP, rep 2; Mus musculus; ChIP-Seq
Cells were cross-linked in PBS containing 2% (v/v) formaldehyde and 10% FBS (v/v) for 10 min at room temperature before quenching with 0.65 M glycine. After washing with ice-cold PBS containing 5% (w/v) bovine serum albumin (BSA), cells were lysed in nuclear extraction buffer (50 mM Tris-HCl [pH7.5], 150 mM NaCl, 5 mM EDTA [pH8], 0.5% NP-40, 1% Triton X-100 in H2O) for 5 min at 4ºC. The released nuclear contents were pelleted and resuspended in sonication buffer (20 mM Tris-HCl [pH7.5], 150 mM NaCl, 2 mM EDTA [pH8], 1% NP-40, 0.3% SDS in H2O). Nuclear fractions were sonicated (peak power: 105 W; duty factor: 20%; cycles per burst: 200) for 10 minutes at 4ºC with a Covaris ultrasonicator (Covaris) to obtain DNA fragments of 100-200 bp. Nuclear debris was removed by centrifugation and the cleared supernatant was collected. 1% (v/v) supernatant was set aside as input control and the remaining supernatant was used for ChIP. ChIP was performed with 2 μg anti-IKZF1 (Santa Cruz Biotechnology) in ChIP dilution buffer (20 mM Tris-HCl [pH8], 150 mM NaCl, 2 mM EDTA, 1% Triton X-100 and 1X cOmplete Mini EDTA-free Protease Inhibitor Cocktail (PIC; Roche Diagnostics) for at least 12 hr at 4°C. Immunoprecipitated lysates were incubated with DynaBeads Protein A/G (Thermo Fisher Scientific) for 2 h at 4°C. Bead/antibody/DNA complexes were washed successively in low salt wash buffer (20 mM Tris-HCl [pH8], 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.15% SDS in H2O), high salt wash buffer (20 mM Tris-HCl [pH8], 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.10% SDS in H2O), LiCl Wash Buffer (20 mM Tris-HCl [pH8], 250 mM LiCl, 2 mM EDTA, 1% Triton X-100, 0.10% (w/v) sodium deoxycholate in H2O) and TE Buffer. ChIP DNA elution was achieved by incubating the washed bead/antibody/DNA complexes in elution buffer (100mM NaHCO3 and 1% SDS in H2O) for 45 min at 45°C and harvesting the supernatant after separation with a DynaMag-2 magnet (Thermo Fisher Scientific). Reverse cross-linking of antibody/DNA complexes in the supernatant was achieved after two successive incubations; first with 5M NaCl for at least 12 h at 65°C then with reverse cross-linking buffer (100 mM NaHCO3, 200 mM NaCl, 1% SDS, 0.3 mg/ml Proteinase K in H2O) for 1 h at 55°C. Free DNA obtained after reverse cross-linking was purified using a ChIP DNA Clean and Concentrator Kit (Zymo Research) according to the manufacturer's protocol. Libraries were constructed using the TruSeq ChIP Library Preparation Kit according to the manufacturer's guidelines