GSM5009730: ChIP-seq_BE2C-QC6352-input-002; Homo sapiens; ChIP-Seq
BE2C 1 x 10e7 of  cells treated with DMSO control or 200 nM of QC6352 for 48 h were cross-linked with freshly prepared 1% formaldehyde (final concentration) for 10 min and quenched with 125 mM glycine (final concentration) for 5 min at room temperature. Cells were washed once with Dulbecco's Phosphate-Buffered Saline (DPBS). 5-8 ml of ice-cold cell lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl and 0.5% NP-40 supplemented with 1 tablet protease inhibitor cocktail) were added to cells. The cells were recovered with a cell scraper and transferred to 50-ml conical tubes on ice. Cell pellets were collected by centrifugation at 2,000 rpm for 5 minutes at 4 °C. The pellets were resuspended in cell lysis buffer and incubated on ice for 10 min. Cells were passed 20 times through a 20-gauge needle. The nuclei were collected by centrifugation at 2,000 rpm at 4 °C for 5 minutes. The nuclear pellets were resuspended and lysed with 250 μl RIPA buffer (1X PBS, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS with 1 tablet protease inhibitor cocktail). The lysates were sonicated for at least 30 min (30 sec on/30 sec off) using Bioruptor Pico (Diagenode, Inc., Denville, NJ) and centrifuged at 14,000 rpm in a microfuge for 15 minutes at 4 °C. The supernatant was collected, and the sonicated DNA fragments were examined by electrophoresis on a 1% agarose gel. The sonicated samples with enrichment of fragments between 100 to 500 bp range were used for the chromatin immunoprecipitation (ChIP) after removing 50 μL from each sample as input. The primary antibodies H3K36me3 (RevMab, 31-1051-00), H3K4me1 (Abcam, ab8895) and H3K27ac (Abcam, ab4729) were coupled with 25 μL magnetic beads (Dynabeads M-280 Sheep Anti-Rabbit IgG, Invitrogen 11203D) and H3K9me3 (Absolute antibody, 309M3-B ) was coupled with 100 μL magnetic beads (Dynabeads M280 Streptavidin, Invitrogen 11205D)  overnight in the cold room at 4 °C, washed three times with PBS/BSA (1X PBS/5 mg/ml BSA (fraction V)) at 4°C, then incubated with sonicated DNA chromatin samples overnight in the cold room at 4°C. The beads containing immuno-bound chromatin were collected by placing the microfuge tube on a magnet rack. The beads were extensively washed with LiCl washing buffer (100 mM Tris pH 7.5/500 mM LiCl/1% NP-40/1% sodium deoxycholate) 5 times and TE buffer (10 mM Tris-HCl pH 7.5 and 0.1 mM Na2EDTA) once. Bound chromatin was recovered by IP elution buffer (1% SDS and 0.1 M NaHCO3) and reverse-crosslinked at 65°C overnight. DNAs were purified using the Mini-Elute PCR Purification Kit (Qiagen, Valencia, CA) after treatment with RNase A and proteinase K. ChIP enriched DNAs were submitted for library preparation and sequencing. 5-10 ng of DNA was using to prepare libraries by the NEBNext ChIP-Seq Library Prep Reagent Set for Illumina with NEBNext Q5 Hot Start HiFi PCR Master Mix according to the manufacturer's instructions (New England Biolabs). Completed ChIP-seq libraries were analyzed for insert size distribution on a 2100 BioAnalyzer High Sensitivity kit (Agilent) or Caliper LabChip GX DNA High Sensitivity Reagent Kit (Perkin Elmer). All libraries were quantified using the Quant-iT PicoGreen dsDNA assay (Life Technologies), Kapa Library Quantification kit (Kapa Biosystems).