GSM4307816: 24hr DMSO A H3K27ac; Homo sapiens; ChIP-Seq
Cells were resuspended in Buffer 1 (50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 0.2% aprotinin, 1 mM PMSF) and rocked at 4°C for 10 min. Samples were centrifuged and resuspended in Buffer 2 (10 mM Tris pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.2% aprotinin, 1 mM PMSF) then rocked at 4°C for 5 min. Samples were centrifuged then resuspended in Buffer 3 (10 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na deoxycholate, 0.5% sarkosyl, 0.2% aprotinin, 1 mM PMSF, Complete mini protease inhibitor cocktail). Samples were sonicated using a Bioruptor (Diagenode) on high setting for 25 cycles (30 sec on, 30 sec off). Next, 1% Triton X-100 was added, and samples were vortexed for 10 sec. Debris was pelleted by centrifuging at 20,000 xg for 10 min, and cleared lysates were transferred to new tubes. At this point, aliquots of chromatin were removed to serve as input controls. One quarter of each sample volume (corresponding to ~10 million cells equivalent) was used for immunoprecipitation. Samples were incubated overnight with 10 μg anti-H3K27ac. Protein A and Protein G Dynabeads were mixed at 1:1 ratio and washed with equilibration buffer (10 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, 1% Triton X-100). Samples were rotated at 4°C for 1 hour after adding 60 μl Dynabeads mix per sample. Beads were washed four times (once in each buffer) by rotating for 5 min at 4°C in the following buffers: low salt wash (20 mM Tris pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100), high salt wash (20 mM Tris pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100), LiCl wash (10 mM Tris pH 8.0, 25 mM LiCl, 1 mM EDTA, 1% Triton X-100), TE. Beads were washed a second time in TE by rotating for 5 min at room temperature. Beads were resuspended in 50 μl elution buffer (TE with 0.1% SDS, 200 mM NaCl, 0.8 μg/μl Proteinase K). Agencourt AMPure XP Beads were used at 1.8X volume according to manufacturer's instructions for size selection. Libraries were preparing by following manufacturer's instructions for NEBNext Ultra II DNA Library Prep Kit for Illumina. Immediately prior to crosslinking, drosophila S2 cells were added to treated THP-1 cells to serve as spike-in control (~1:27 ratio of drosophila:human cells). Cells were crosslinked with 1% formaldehyde, quenched with glycine, and washed with cold PBS. See "ChIP protocol" column above for experiment-specific protocols.