GSM6482634: H3K27ac_Pgc1b_flox_Ad_RFP_Day4 rep2; Mus musculus; ChIP-Seq
Chromatin immunoprecipitation (ChIP) was performed in biological replicates as previously described (Heinz et al., 2018: Sakai et al., 2019) with modifications as follows.  For H3K27ac ChIP assays, cells were cross-linked with 1% (vol/vol) formaldehyde (Thermo Fisher Scientific) in PBS for 10 min at room temperature.  For NCoR1, HDAC3, PU.1, p65, Jdp2, Fosl2, PGC1b and FLAG-tag ChIP assays, cells were cross-linked with 2 mM disuccinimidyl glutarate (DSG) (ProteoChem) in PBS for 30 min at room temperature, and then directly a second crosslinking was performed by the addition of 1% (vol/vol) formaldehyde in PBS for 10 min.  The crosslinking reaction was quenched by 0.125 M glycine (Thermo Fisher Scientific).  Cells were washed once with cold PBS and pelleted at 1,000 g for 5 min at 4°C.  Crosslinked cells were resuspended in ice-cold hypotonic buffer (10 mM HEPES-KOH (pH 7.9), 85 mM KCl, 1 mM EDTA, 1% NP-40, 1 mM PMSF (Sigma), 0.5 mM sodium butyrate (Sigma), 1X protease inhibitor cocktail (Sigma)), and centrifuged at 1,000 g for 5 min at 4°C to obtain a nuclear fraction.  Nuclear pellets were resuspended in 100 ml of either LB3 buffer (10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 0.5% N-laurosylsarcosine, 1 mM PMSF, 0.5 mM sodium butyrate, 1X protease inhibitor cocktail for H3K27ac ChIP) or PIPA-NR buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.4% sodium deoxycholate, 0.1% SDS, 1% NP-40, 0.5 mM DTT, 1 mM PMSF, 0.5 mM sodium butyrate, 1X protease inhibitor cocktail, for NCoR, HDAC3, PU.1, p65, Fosl2, PGC1b and FLAG-tag ChIP).  Chromatin DNA was sonicated in a 96 Place microTUBE Rack (Covaris) using a Covaris E220 for 12-18 cycles 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 centrifuged at 15,000 rpm for 10 min at 4°C, and the supernatant was used for immunoprecipitation.  LB3 lysate was diluted 1.1-fold with 10% Triton X-100.  1% of the lysate was kept as ChIP input.  The lysates were rotated with each antibody pre-bound to 20 ml of beads (10 ml of Dynabeads protein A (Invitrogen) + 10 ml of Dynabeads protein G (Invitrogen)) overnight at 4°C.  After the immunoprecipitation, 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, 1 mM PMSF, 0.5 mM sodium butyrate, 1X protease inhibitor cocktail), wash buffer III (10 mM Tris-HCl (pH 7.5), 250 mM LiCl, 1% Triton X-100, 0.7% sodium deoxycholate, 1 mM EDTA, 1 mM PMSF, 0.5 mM sodium butyrate, 1X protease inhibitor cocktail) and twice with TET (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.2% Tween-20, 1 mM PMSF, 0.5 mM sodium butyrate, 1X protease inhibitor cocktail), and then resuspended in 25 ml of TT (10mM Tris-HCl (pH 7.5), 0.05% Tween-20). The immunoprecipitated chromatin samples were used for library preparation with NEBNext Ultra II Library kit (NEB) according to the manufacturer's instructions.  DNA in 46.5 ml of NEB reactions was crosslinks reversed by adding 4 ml of 10% SDS, 4.5 ml of 5 M NaCl, 3 ml of 500 mM EDTA, 1 ml of 20 mg/ml proteinase K (NEB) and 20 ml of water by incubation for 1 hr at 55°C, and then 30 min at 65°C.  DNA was purified with 2 ml of SpeedBeads (GE Healthcare) in 20% PEG 8000/1.5 M NaCl (Final 12% PEG 8000), and eluted with 25 ml of TT.  The eluted DNA was amplified for 14 cycles in 50 ml of PCR reactions using NEBNext High-Fidelity 2X PCR Master Mix and 0.5 mM each of primers Solexa 1GA and Solexa 1GB.  The amplified libraries were purified with 2 ml of SpeedBeads in 20% PEG 8000/2.5 M NaCl (Final 13% PEG 8000), eluted with 20 ml of TT, size selected using PAGE/TBE gel (Invitrogen) for 225-325 bp fragments by gel extraction, and single-end sequenced on HiSeq 4000.  ChIP input material (1% of sheared DNA) in 46.5 ml of TE was crosslinks reversed by adding 4 ml of 10% SDS, 4.5 ml of 5 M NaCl, 3 ml of 500 mM EDTA, 1 ml of 20 mg/ml proteinase K and 20 ml of water by incubation for 1 hr at 55°C, and then 30 min at 65°C.  The input DNA was purified with SpeedBeads as described above, and eluted with 25 ml of TT.  The eluted input DNA was prepared for libraries and amplified as described for ChIP samples.