title: GSM6736160: BV173 + IDASA p53 ChiP; Homo sapiens; ChIP-Seq
library_construction_protocol: 1 x 10^7 BV173 cells were pelleted by centrifugation and resuspended in FBS-free RPMI media and cross-linked by adding methanol-free paraformaldehyde (VWR; final conc. 1%) for 15 min with gentle stirring. Cross-linking was quenched by adding glycine [0.125M]. Cells were then pelleted by centrifugation, washed in PBS and pelleted again. Cells were resuspended in ∼1.5 x pellet volume of cell lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA-630; protease inhibitors) and incubated for 10 min on ice. The cell nuclei were pelleted by centrifugation at 2500 rpm for 5 min at 4°C and resuspended in 1.2 mL of nuclear lysis buffer (NLB 50 mM Tris-HCl pH 8.1, 10 mM EDTA, 1% SDS, protease inhibitors) and incubated on ice for 10 min. Volume was increased to 2 mL by adding 0.72 mL of immunoprecipitation (IP) dilution buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA 1% Triton X-100, 0.01% SDS, protease inhibitors) and the chromatin was transferred to a 5 mL tube (Falcon) and sheared to a fragment size of 200-500 bp by sonication (Soniprep 150, MSE). The sheared chromatin was diluted with 4.1 mL of IP dilution buffer and pre-cleared by adding 10 μg rabbit IgG (12-370, Millipore) for 1 hr followed by incubation with 200 μL of protein G-agarose bead suspension (Roche) for 3 hr. The protein G-agarose beads were removed by brief centrifugation and 1.35 mL of the chromatin was added to 10 μg of p53 antibody (DO-1, sc-126, Santa Cruz Biotechnology) for overnight incubation. ChIP was carried out by adding 100 μL protein G-agarose beads suspension for 3 hr. Beads were washed (twice in 20 mM Tris-HCl pH 8.1, 50 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.01% SDS; once in 10 mM Tris-HCl pH 8.1, 250 mM LiCl, 1 mM EDTA, 1% IGEPAL CA630, 1% deoxycholic acid; twice in 10 mM Tris-HCl 1 mM EDTA pH 8.0). The immune complexes were eluted from the beads (twice in 225 μL of 100 mM NaHCO3, 0.1% SDS). Crosslinks were reversed by adding 0.2 μL of RNase A (10 mg/mL, ICN) and 27 μL of 5 M NaCl to the eluates with incubation at 65°C for 6 hr and then overnight at 45oC in the presence of 90 μg proteinase K (Invitrogen). DNA was purified by phenol chloroform extraction followed by ethanol precipitation and resuspension in 50 μL of water. ChIP-sequencing libraries were generated using the Nextera XT DNA Library Preparation Kit (Illumina) with dual indexing (Nextera XT Index Kit, Illumina) following manufacturers' instructions.  Libraries were quantified using the Agilent Bioanalyzer High Sensitivity DNA Kit.