GSM6504832: BMDM_pbs_input_i5; Mus musculus; ChIP-Seq
30 million BMDMs were crosslinked in 2mM Disuccinimmidyl Glutarate (Pierce) and 1% formaldehyde. BMDMs were resuspended in swelling buffer (10 mM HEPES/KOH pH7.9, 85 mM KCl, 1 mM EDTA, 0.5% IGEPAL CA-630, 1x protease inhibitor cocktail, 1 mM PMSF) and incubated on ice for 5 minutes. Cells were spun down and lysed in 130 µl RLNR1 lysis buffer (20 mM Tris/HCl pH7.5, 1 mM EDTA, 0.5 mM EGTA, 0.1% SDS, 0.4% Na-Deoxycholate, 1% NP-40 alternative, 0.5 mM DTT, 1x protease inhibitor cocktail, 1 mM PMSF) and chromatin was sheared by sonication in a 96 Place microTUBE Rack (Covaris cat#500282) using a Covaris E220 for 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 recovered and spun down at max speed, 4°C for 10 minutes. 1% supernatant was taken as input DNA and remaining supernatant was transferred to PCR strips and brought up to a volume of 200 µl using RLNR1 lysis buffer (20 mM Tris/HCl pH7.5, 1 mM EDTA, 0.5 mM EGTA, 0.1% SDS, 0.4% Na-Deoxycholate, 1% NP-40 alternative, 0.5 mM DTT, 1x protease inhibitor cocktail, 1 mM PMSF). 30 µl Dynabeads Protein A/G coated with the indicated antibodies for SREBP1/2 (antibody details in Supplementary Table 1) were added to the sample, and immunoprecipitation was carried out at 4°C overnight. Beads were then collected using a magnet and washed with 175 µl ice-cold buffer as indicated by incubating samples on ice for 3 minutes: 3 times RLNR1 lysis buffer (20 mM Tris/HCl pH7.5, 1 mM EDTA, 0.5 mM EGTA, 0.1% SDS, 0.4% Na-Deoxycholate, 1% NP-40 alternative, 0.5 mM DTT, 1x protease inhibitor cocktail, 1 mM PMSF), 6 times LiCl-NR1 buffer (10 mM Tris/HCl pH7.5, 250 mM LiCl, 1 mM EDTA, 0.7% Na-Deoxycholate, 1% NP-40 alternative, 1x protease inhibitor cocktail, 1 mM PMSF), 3 times TET (10 mM Tris/HCl pH 8.0, 1 mM EDTA, 0.2% Tween-20, 1x protease inhibitor cocktail, 1 mM PMSF), and 1 time IDTET (10 mM Tris/HCl pH 8.0, 0.1 mM EDTA, 0.2% Tween-20, 1x protease inhibitor cocktail, 1 mM PMSF). 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 DNA Library preparation kit. 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 5 M NaCl, 3 µl 0.5 M EDTA, and 1 µl 20 mg/ml proteinase K, 1 µl RNase E, followed by incubation at 55°C for 1 hour and 65°C for 2 hours 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 RT 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 ethanol wash, beads were air-dried and DNA eluted in 13 µl 10 mM Tris/HCl pH 8.0 and 0.05% Tween-20. DNA was amplified by PCR for 14 cycles in a 25 µl reaction volume using NEBNext Ultra II PCR master mix and 0.5 µM each Solexa 1GA and Solexa 1GB primers. PCR amplified libraries were size selected 200-500 bp using gel extraction using 10% TBE acrylamide gels.