GSM3772751: J1.Unt_GABPA_REP3; Mus musculus; ChIP-Seq
Chromatin was prepared from 1 x 108 mouse ES cells grown to 70-90% confluency. Cells were crosslinked at room temperature for either 10 min in 1% methanol-free formaldehyde (Thermo Scientific) or 2 mM disuccinimidyl glutarate (DSG) for 1 hour followed by 10 min with 1% formaldehyde (see "description"). Crosslinking reactions were quenched by the addition of glycine to a concentration of 200 mM. Chromatin was prepared at 4 °C by rotating the cells in 10 ml buffer LB1 (50 mM HEPES KOH pH 7.9, 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP40, 0.25% Triton x-100, with 1x cOmpleteTM EDTA-free Protease Inhibitor Cocktail (Roche)), 10 ml buffer LB2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, with 1x cOmpleteTM EDTA-free Protease Inhibitor Cocktail) and 1 ml buffer LB3 (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine, 1x cOmpleteTM EDTA-free Protease Inhibitor Cocktail). Chromatin was sheared in a 1 ml milliTUBE with AFA Fiber (Covaris) using a Covaris S220 AFA ultrasonicator (set at 150 W Peak Incident Power, 8% Duty Factor, 200 Cycles/Burst) for 30 min. Following sonication, the sheared chromatin was spun at 15,000 x g for 20 min and the soluble fraction collected and aliquoted.  Immunoprecipitation (IP) was performed using the sheared chromatin from approximately 1 x 107 cells in Chromatin Dilution Buffer (16.7 mM Tris pH 8.1, 167 mM NaCl, 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, freshly complemented with 1x cOmpleteTM EDTA-free Protease Inhibitor Cocktail). Following a 30 min preclear using 50 µl blocked Protein A or G agarose beads (Roche), the chromatin was incubated with 5 µg antibody overnight at 4 °C. Immune complexes were captured with blocked Protein A or G agarose beads by incubation for 4 h with rotation and then washed with the following buffers: Low Salt Buffer (20 mM Tris pH8.1, 150 mM NaCl, 0.2% SDS, 1% Triton X-100, 5 mM EDTA, and 5% w/v sucrose), High Salt Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 0.1% deoxycholic acid, 1% Triton X-100 and 1 mM EDTA), Lithium Chloride Buffer (10 mM Tris pH 8.0, 250 mM LiCl, 0.5% deoxycholic acid, 0.5% NP-40 and 1 mM EDTA) and twice with TE Buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Captured chromatin was eluted in 125 µl elution buffer (1% SDS and 0.1 M NaHCO3) by shaking vigorously for 30 min at room temperature and then subjected to reverse crosslinking by incubating at 65 °C overnight in the presence of 200 mM NaCl. Input controls consisted of 50 µl (5%) samples of pre-cleared chromatin that were reverse crosslinked and purified as described for the IP samples. After incubation with 0.5 µg RNase A (1 h at 37 °C) and 20 µg Proteinase K (1 h at 45 °C), DNA was purified using the QIAquick PCR Purification kit (Qiagen). Automated library preparation was performed on 5 ng input material using the Apollo prep system (Wafergen, PrepX ILMN 32i, 96 sample kit) and standard Illumina multiplexing adapters following manufacturer's protocol up to pre-PCR amplification. Libraries were PCR amplified (18 cycles) on a Tetrad (Bio-Rad) using the NEBNext High-Fidelity 2X PCR Master Mix (NEB) and in-house single indexing primers. Equivalent amounts of individual libraries were pooled together. Paired-end sequencing was performed using a HiSeq4000 75 bp platform (Illumina, HiSeq 3000/4000 PE Cluster Kit and 150 cycle SBS Kit), generating a raw read count of >30 million reads per sample.