GSM8641348: DMSO_H3K9acOH; Homo sapiens; ChIP-Seq
Chromatin-bound proteins were crosslinked to DNA by addition of 37% formaldehyde to a concentration of 1% (w/v) followed by gentle rocking on ice (10 min). The reaction was quenched by addition of glycine (125 mM) solution and followed by gentle rocking at room temperature (10 min). Cells were washed twice with PBS before being physically scraped into 5 mL PBS. This suspension was centrifuged (4°C, 800 rpm, 10 min). The supernatant was discarded and pelleted cells were then resuspended in 500 µL of ChIP SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris pH 8.1, supplemented with fresh Roche complete protease inhibitor cocktail (2× concentration)) before incubation on ice (10 min). Lysates were diluted with ChIP dilution buffer (500 µL, 0.01% SDS, 1.1% Triton™ X-100, 1.2 mM EDTA, 16.7 mM Tris pH 8.1, 167 mM NaCl) and transferred to Bioruptor Plus TPX tubes suitable for sonication. Chromatin fragmentation was accomplished by 30 minutes of sonication using a Bioruptor ® Plus set to 'HIGH' mode (15 s on / 15 s off). The sonicated chromatin was centrifuged (13,000 rpm, 4 °C, 10 min), the supernatant was collected. A 20 µL aliquot of sonicated chromatin was taken for quantification and the rest snap-frozen for storage at -80°C. Fresh elution buffer (220 µL, 0.1 M NaHCO3, 1% SDS), was added to an aliquot and NaCl solution (12.5 µL, 4 M) was added to each sample and shaken overnight on an Eppendorf ThermoMixer F1.5 set to 65°C, 1400 rpm. Proteins were digested with Proteinase K (Thermo Fisher, 2 µL) and shaken (1400 rpm, 45°C, 4 h). For RNA degradation, RNase A (Thermo Fisher, 1.0 µL) was added and shaken (1400 rpm, 37 °C, 30 min). Samples were then mixed with PB binding buffer (Qiagen, 1.0 mL) and sodium acetate solution (10 µL, 3 M, pH 5.2). DNA was purified with MinElute PCR Purification columns (Qiagen) and eluted with nuclease-free water (20 µL). Chromatin fragmentation was assessed by Agilent 2200 TapeStation System Automated Electrophoresis and D1000 DNA ScreenTape analysis and ~200 bp was considered acceptable. The concentration of purified DNA was quantified by Qubit 2.0 fluorometer (Invitrogen) and dsDNA Broad Range Assay kit (Thermo Fisher). A pre-determined amount of chromatin (detailed in Supplementary Table 5) was taken from each sample and diluted with ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris, 167 mM NaCl, pH 8.1) to achieve a final volume of 1.0 mL in 2.0 mL Eppendorf DNA LoBind safe-lock microcentrifuge tubes (Thermo Fisher). This fixed amount of Human chromatin was combined with an antibody of interest according to the details in Supplementary Table 5. Drosophila melanogaster chromatin (Active Motif, 10 ng) and Drosophila-specific histone variant H2Av antibody (Active Motif, 1 µg) was added to each reaction as a spike-in control for downstream normalization. For ChIP-Seq determining H3K9acOH, the Drosophila chromatin (5 ng) and antibody (0.5 µg) was reduced. These chromatin/antibody reactions were rotated overnight (4 °C), antibody-bound DNA was pulled down by magnetic Protein G Dynabeads (Thermo Fisher). Beads (50 µL) were added to each sample along with ChIP dilution buffer (500 µL) and incubated on the end-over-end rotator (4 °C, 2 h). After the supernatant was removed by utilizing a magnetic rack, the samples were washed using low salt wash buffer (500 µL, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8.1, 150 mM NaCl), high salt wash buffer (500 µL, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8.1, 500 mM NaCl), LiCl wash buffer (500 µL, 1% Igepal, 1 mM EDTA, 10 mM Tris, 250 mM LiCl, 1% sodium deoxycholate, pH 8.1), and two rounds of TE wash (500 µL, 10 mM, 1 mM EDTA, Tris pH 8.0) using sample rotary machine (4°C, 5 min). Fresh elution buffer was added to the beads (120 µL) and shaken (1400 rpm, room temperature, 15 min). The supernatant was collected; the elution process was repeated and the eluates were pooled. All libraries were prepared and sequenced (single-end) by the US NCI CCR Genomics Core following Illumina recommended protocols for the NextSeq 500 75 cycle High output platform.