GSM9045470: HNSCC_Detroit562_Parental_ChIPseq_H3K27ac; Homo sapiens; ChIP-Seq
For ChIP-Seq of post-translation modifications (PTMs), approximately 20 million cells per cell line were used. Crosslinking was performed in 150 mm cell culture plates using 1% formaldehyde (Sigma) at room temperature with gentle rocking for 10 minutes. The crosslinking reaction was quenched using 1.25 M Glycine with gentle rocking at room temperature for 5 minutes. Fixed cell preparations were washed with ice-cold PBS, scraped, and then washed twice more with ice-cold PBS. Crosslinked pellets were resuspended in 500 μl cell lysis buffer (5 mM pH 8.5 PIPES, 85 mM KCl, 1% (v/v) IGEPAL CA-630, 50 mM NaF, 1 mM PMSF, 1 mM phenylarsine oxide, 5 mM sodium orthovanadate, EDTA-free protease inhibitor tablet) and incubated for 30 minutes on ice. Samples were centrifuged and pellets were resuspended in 500 μl of nuclei lysis buffer (50 mM pH 8.0 Tris-HCl, 10 mM EDTA, 1% (w/v) SDS, 50 mM NaF, 1 mM PMSF, 1 mM phenylarsine oxide, 5 mM sodium orthovanadate, EDTA-free protease inhibitor tablet) and incubated for 30 minutes on ice. Sonication of lysed nuclei was performed using the BioRuptor UCS-300 at maximum intensity for 75-90 cycles (10s on, 20s breaks). Sonication efficiency to achieve fragments between 150 - 500 bp was evaluated using gel electrophoresis on a reversed-crosslinked and purified aliquot from each sample. After sonication, chromatin was diluted to reduce SDS levels to 0.1% and concentrated using Nanosep 10K OMEGA (Pall) columns. Prior to the chromatin immunoprecipitation (ChIP) reaction, 2% sonicated Drosophila S2 cell chromatin was spiked into each sample for quantification of total levels of histone marks. The ChIP reactions were performed using the Diagenode SX-8G IP-Star Compact and Diagenode automated iDeal ChIP-seq Kit for Histones. Dynabeads Protein A (Invitrogen) were washed, then incubated with specific antibodies, 1.5 million cells of sonicated cell lysate, and protease inhibitors for 10 hrs, followed by a 20 min wash cycle using the provided wash buffers (Diagenode Immunoprecipitation Buffers, iDeal ChIP-seq Kit for Histones). Reverse cross-linking was then performed using 5M NaCl at 65℃ for 4 hrs. ChIP samples were then treated with 2 μl RNase Cocktail at 65℃ for 30 min followed by 2 μl Proteinase K at 65℃ for 30 min. Samples were then purified with QIAGEN MinElute PCR purification kit (QIAGEN) as per the manufacturer's protocol. In parallel, input samples (chromatin from about 50,000 cells) were reverse crosslinked and DNA was isolated following the same protocol. For protein ChIP-Seq (i.e. HP1), Approximately 40 million cells per cell line were used. Crosslinking was performed in 150 mm cell culture plates using 1% formaldehyde (Sigma) at room temperature with gentle rocking for 10 minutes. The crosslinking reaction was quenched using 1.25 M Glycine with gentle rocking at room temperature for 5 minutes. Fixed cell preparations were washed with ice-cold PBS, scraped, and then washed twice more with ice-cold PBS. Crosslinked pellets were resuspended in 500 μl cell lysis buffer (5 mM pH 8.5 PIPES, 85 mM KCl, 1% (v/v) IGEPAL CA-630, 50 mM NaF, 1 mM PMSF, 1 mM phenylarsine oxide, 5 mM sodium orthovanadate, EDTA-free protease inhibitor tablet) and incubated for 30 minutes on ice. Samples were centrifuged and pellets were resuspended in 500 μl of nuclei lysis buffer (50 mM pH 8.0 Tris-HCl, 10 mM EDTA, 1% (w/v) SDS, 50 mM NaF, 1 mM PMSF, 1 mM phenylarsine oxide, 5 mM sodium orthovanadate, EDTA-free protease inhibitor tablet) and incubated for 30 minutes on ice. Sonication of lysed nuclei was performed using the BioRuptor UCS-300 at maximum intensity for 75-90 cycles (10s on, 20s breaks). Sonication efficiency to achieve fragments between 150 - 500 bp was evaluated using gel electrophoresis on a reversed-crosslinked and purified aliquot from each sample. After sonication, chromatin was diluted to reduce SDS levels to 0.1% and concentrated using Nanosep 10K OMEGA (Pall) columns. Prior to the chromatin immunoprecipitation (ChIP) reaction, 2% sonicated Drosophila S2 cell chromatin was spiked into each sample for quantification. For each IP, Dynabeads Protein A or G (Invitrogen) were washed with RIPA buffer and then resuspended in RIPA buffer. Specific antibodies were added (Supplementary Table 2) and then incubated at 4℃ for at least 3 hours on a rotator. Following antibody conjugation, Dynabeads were washed with RIPA buffer, and then resuspended in RIPA buffer, ~5 million cells of sonicated cell lysate, and protease inhibitors, followed by overnight incubation on a rotator at 4℃. Four successive washes were then performed using 1. RIPA buffer; 2. RIPA buffer with 500 mM NaCl (1% (v/v) Triton X-100, 0.10% SDS, 500 mM NaCl, 20 mM Tris-HCl pH 8.0, 2 mM EDTA, H2O); 3. LiCl buffer (1% NP-40, 250 mM LiCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, H2O); 4. TE buffer (50 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, H2O). Samples were then resuspended in Elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% (v/v) SDS, H2O) and incubated at room temperature for 30 minutes. Reverse cross-linking was then performed using 5M NaCl at 65℃ for 4 hrs. ChIP samples were then treated with 2 μl RNase Cocktail at 65℃ for 30 min followed by 2 μl Proteinase K at 65℃ for 30 min. Samples were then purified with QIAGEN MinElute PCR purification kit (QIAGEN) as per the manufacturer's protocol. In parallel, input samples (chromatin from about 50,000 cells) were reverse crosslinked and DNA was isolated following the same protocol. Library preparation was performed using the Kapa Hyper Prep library preparation reagents following the manufacturer's protocol (Kapa Hyper Prep Kit, Roche 07962363001). Library preparation was performed using the Kapa Hyper Prep library preparation reagents following the manufacturer's protocol (Kapa Hyper Prep Kit, Roche 07962363001). ChIP libraries were sequenced by the McGill University Genome Centre using the Illumina HiSeq 4000 at 50 bp single reads.