title: GSM3445663: SMARCA2-infected H1703 H3K27Ac ChIP; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were fixed in complete media with 1% formaldehyde for 10 minutes at room temperature or 0.3% formaldehyde for 30 minutes at 4ºC then quenched by addition of 0.125 M glycine for 5 minutes at room temperature and 15 minutes on ice. Fixed cells were then pelleted and washed once with 1x PBS before snap-freezing on dry-ice. Cell pellets were lysed in three successive buffers for 10 minutes each at 4ºC while rotating end-over-end (LB1: 50 mM HEPES-KOH pH 7.5, 120 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, LB2: 10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, LB3: 10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine). Lysates were then sonicated with a Branson450D cup-horn system to produce chromatin fragments between 100 and 600 bps. Triton X-100 was added to cell lysate and then centrifuged at 20,000 g for 15 minutes at 4ºC to pellet debris. Supernatant equivalent to 10 million cells brought to a final volume of 500 µL using LB3 was used for each immunoprecipitation. A quantity of sonicated chromatin was set aside as 10% input. 2 µg of Histone H3 acetyl K27 (abcam, ab4729) or Histone H3 tri-methyl K4 (abcam, ab8580) antibodies were added to the lysate for overnight incubation at 4ºC. Protein G Magnetic Dynabeads® (ThermoFisher Scientific) were used for pulldown. Immunoprecipitated chromatin bound to dynabeads was washed with four successive buffers (LSB: 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA; MSB: 20 mM Tris-HCl pH 8.0, 250 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA; LiCl wash: 10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate, 1 mM EDTA; 1x TE: 10 mM Tris-HCl pH 8.0, 1 mM EDTA). Chromatin was then eluted from dynabeads in 150 µL EB (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS) by incubating at 65ºC for 30 minutes. Immunoprecipitated samples and input were incubated overnight at 65ºC to denature formaldehyde crosslinking. Samples were then treated with RNaseA (ThermoFisher Scientific) followed by proteinase K (Sigma Aldrich) before phenol/chloroform extraction. DNA was precipitated using 5M NaCl, glycoblue (Ambion) and 100% absolute ethanol overnight at -80ºC. DNA was pelleted by centrifuging at 20,000 g for 30 minutes at 4ºC followed by a 70% ethanol wash. Final DNA pellet was resuspended in 50 uL of 1x TE buffer and placed in speedvac for 3 minutes. ChIP-seq libraries were generated using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs).
attributes.tag: GEO Accession
attributes.value: GSM3445663