title: GSM3681863: ChIP_H3K27ac_KC_notx_rep1; Mus musculus; ChIP-Seq
library_construction_protocol: For H3K27ac ChIP-seq, culture media was removed and plates were washed once with PBS and then fixed with 1% formaldehyde (Thermo Fisher Scientific) in PBS for 10 minutes at room temperature. The reaction was quenched by adding glycine (Thermo Fisher Scientific) to 0.125M. For LXR, SMAD4, and RBPJ ChIP-seq, cells were crosslinked with 3mM DSG (ProteoChem) in PBS for 30 minutes at room temperature, and then fixed with 1% formaldehyde (Thermo Fisher Scientific) in PBS for 10 minutes at room temperature. The reaction was quenched by adding glycine (Thermo Fisher Scientific) to 0.125M. After fixation, cells were washed once with cold PBS and then pelleted at 700 X G for 5 minutes at 4°C. Crosslinked cells were stored at –80°C until ready for ChIP-seq library preparation. Fixed cells were thawed on ice, resuspended in either ice-cold LB3 (10 mM Tris/HCl pH 7.5, 100 mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine, 1 X protease inhibitor cocktail, 1mM Na-Butyrate, for H3K27ac ChIP) or ice-cold RIPA-NR lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.4% Na-Deoxycholate, 0.1% SDS, 1% NP-40, 1x protease inhibitors, for LXR, SMAD4, and RBPJ ChIP). Frozen crosslinked nuclei were resuspended in wash buffer (10mM HEPES/KOH pH7.9, 85mM KCl, 1mM EDTA, 0.2% IGEPAL CA-630, 1x protease inhibitor cocktail (Sigma), 1 mM PMSF) for 5minutes on ice. Nuclei were spun down and then resuspended in RIPA-NR lysis buffer. Chromatin was sheared by sonication. Samples were sonicated in a 96 Place microTUBE Rack (Covaris cat#500282) using a Covaris E220 for 12 cycles (samples in LB3) or 18 cycles (samples in RIPA-NR buffer) with the following setting: time, 60 seconds; duty, 5.0; PIP, 140; cycles, 200; amplitude, 0.0; velocity, 0.0; dwell, 0.0. Samples were recovered and spun down at max speed, 4°C for 10 minutes. The LB3 lysate was diluted 1.1-fold with ice-cold 10% Triton X-100 after sonication. One percent of the lysate was kept as ChIP input. For each immunoprecipitation, aliquots of diluted lysate equivalent to 500,000 cells (for H3K27ac ChIP) or 2-3 million cells or nuclei (for LXR, SMAD4, or RBPJ ChIP), 30 µl of Dynabeads Protein A (for rabbit polyclonal antibodies) or Dynabeads protein A/G (for LXR ChIP) bound to specific antibodies for H3K27ac (2 μg, Active Motif, 39133), LXR (2 µg each of the indicated LXR specific antibodies, Santa Cruz Biotechnology: sc-1000X, sc-133221X, sc-271064X)), SMAD4 (1 μg each of Cell Signaling technology 46535 and 38454) or RBPJ (2 μg, Abcam, ab25949) were combined and rotated overnight at 4°C. For H3K27ac ChIP, beads were collected on a magnet and washed three times each with wash buffer I (20 mM Tris/HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA), wash buffer III (10 mM Tris/HCl pH 7.4, 250 mM LiCl, 1% Triton X-100, 0.7% Na-Deoxycholate, 1 mM EDTA) and twice with ice-cold TET (10 mM Tris/HCl pH7.5, 1 mM EDTA, 0.2% Tween-20). For LXR, SMAD4, or RBPJ ChIP, beads were washed three times with RIPA-NR buffer (20 mM Tris-HCl/pH7.5, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.4 % Na-Deoxycholate, 1 mM EDTA, 0.5mM EGTA, 0.5mM DTT), six times with RIPA-LiCl buffer (10 mM Tris-HCl/pH 7.5, 250 mM LiCl, 1% NP-40, 0.7% Na-Deoxycholate, 1 mM EDTA), three times with ice-cold TET (10 mM Tris/HCl pH7.5, 1 mM EDTA, 0.2% Tween-20), and one time with IDTE (10mM Tris-HCl pH 8.0, 0.1mM EDTA). Libraries were prepared with NEBNext Ultra II DNA library prep kit (NEB) reagents according to the manufacturer's protocol on the beads suspended in 25 µL TT (10mM Tris/HCl pH7.5, 0.05% Tween-20), with reagent volumes reduced by half. DNA was eluted and crosslinks reversed by adding 4 µl 10% SDS, 4.5 µl 5 M NaCl, 3 µl EDTA, 1 µl proteinase K (20 mg/ml), 20 µl water, incubating for 1 h at 55°C , then 30 minutes to overnight at 65°C . DNA was purified using 2 µL of SpeedBeads (GE Healthcare), diluted with 20% PEG8000, 1.5M NaCl to final of 12% PEG, eluted with 25 µl TT. DNA contained in the eluate was then amplified for 12 cycles in 50 µl PCR reactions using NEBNext High-Fidelity 2X PCR Master Mix (NEB) and 0.5 mM each of primers Solexa 1GA and Solexa 1GB. ChIP input material (1 percent of sheared DNA) was treated with RNase for 15 minutes at 37°C in EB buffer (10 mM Tris pH 8, 0.5% SDS, 5 mM EDTA, 280 mM NaCl), then digested with Proteinase K for 1 h at 55°C and crosslinks reversed at 65°C for 30 minutes to overnight. DNA was purified using 2 µL of SpeedBeads (GE Healthcare), diluted with 20% PEG8000, 1.5M NaCl to final of 12% PEG, eluted with 25 µl TT and library prep and amplification were performed as described for ChIP samples. Resulting libraries were size selected by gel excision to 225-325 bp and purified.
attributes.tag: GEO Accession
attributes.value: GSM3681863