alias: GSM5160131
title: GSM5160131: p300 ChIP-seq  GW3965 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 p300, LXR and SREBP 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 p300, LXR, and SREBP 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. Cells 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 1-2 million cells  (for p300, SREBP ChIP), 20 µl of Dynabeads Protein A (for rabbit polyclonal antibodies) or Dynabeads protein A/G (for p300, LXR, and SREBP ChIP) bound to specific antibodies for H3K27ac (2 μg, Active Motif, 39133), p300 (2 μg, Millipore, cat# 05-257,Diagenode, cat# C15200211), LXR (Santa Cruz Biotechnology: sc-1000X, sc- 133221X, sc-271064X) , and SREBP (2 μg, Santa Cruz Biotechnology: sc-8984X, Thermo Fisher Scientific: PA1-337, R&D Systems: AF7119) 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 p300, LXR and SREBP 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). Bead complexes were resuspended in 25 μl TT (10 mM Tris/HCl pH 8.0, 0.05% Tween-20, 1x protease inhibitor cocktail) and sequencing libraries were prepared on-bead as described below. ChIP libraries were prepared while bound to Dynabeads using NEBNext Ultra II Library preparation kit with reaction volumes reduced by half, essentially as previously described (Heinz et al., 2018). Libraries were eluted and crosslinks reversed by adding to the 46.5 μl NEB reaction 20 μl water, 4 μl 10% SDS, 4.5 μl 5M NaCl, 3 μl 0.5 M EDTA, and 1 μl 20 mg/ml proteinase K, followed by incubation at 55C for 1 hour and 65C for 30 minutes to overnight in a thermal cycler. Dynabeads were removed from the library using a magnet and libraries cleaned by adding 2 μl SpeedBeads 3 EDAC in 61 μl 20% PEG 8000/1.5 M NaCl, mixing well, then incubating at room temperature for 10 minutes. SpeedBeads were collected on a magnet and washed two times with 150 μl 80% ethanol for 30 seconds. Beads were collected and ethanol removed following each wash. After the second ethanol wash, beads were air dried and DNA eluted in 25 μl 10 mM Tris/HCl pH 8.0 and 0.05% Tween-20. DNA was amplified by PCR for 14 cycles in a 50 μl reaction volume using NEBNext Ultra II PCR master mix and 0.5 μM each Solexa 1GA and Solexa 1GB primers. Libraries were cleaned up with SpeedBeads as above using 36.5 μl 20% PEG 8000/1.5 M NaCl and 2 μl SpeedBeads. After ethanol washing and drying, PCR amplified libraries were eluted from the SpeedBeads using 20 μl 10 mM Tris/HCl pH 8.0 and 0.05% Tween- 20. Next, libraries were size selected 250-500 bp using gel extraction using 10% TBE acrylamide gels. Libraries were single-end sequenced using either a HiSeq 4000 or a NextSeq 500 to a depth of 10-20 million reads.
identifiers.namespace: GEO
identifiers.id: GSM5160131
attributes.tag: GEO Accession
attributes.value: GSM5160131