GSM8723249: QGP-1, control, 3, K9Ac; Homo sapiens; ChIP-Seq
QGP-1 cells were trypsinized, crosslinked to 1% formaldehyde for 10 min, and quenched with 125 mM glycine for 5 min at room temperature. After washing with TBS, cells were resuspended in cell lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 0.5% NP-40) and incubated on ice for 10 min. Lysates were washed with MNase digestion buffer (20 mM Tris-HCl, pH 7.5, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2). After resuspension in 200 μL fresh MNase digestion buffer containing proteinase inhibitor cocktail (Sigma), the lysates were incubated with 2000 units of MNase (New England Biolabs, Ipswich, MA) per 4 x 10^6 cells at 37°C for 20 min with continuous mixing in a thermal mixer. After adding 200 μL of sonication buffer (100 mM Tris-HCl, pH 8.1, 20 mM EDTA, 200 mM NaCl, 2% Triton X-100, 0.2% sodium deoxycholate), the lysates were sonicated for 20 min (30 s on/30 s off) using Bioruptor Twin (UCD-400) (Diagenode, Denville, NJ) and centrifuged at 21,130 x g for 10 min. The supernatants were collected, and the chromatin amount was estimated by the Qubit assay (Invitrogen). ChIP-Rx; The chromatin was then incubated with anti-H3K27ac antibody (Cell Signaling Technology, Danvers, MA) or in-house generated anti-H3K9ac antibody (EDL lot 1) on a rocker overnight. Protein G-magnetic beads (30 μL, Life Technologies) were added, and further incubated for 3 hrs. The beads were extensively washed with ChIP buffer (50 mM Tris-HCl, pH 8.1, 10 mM EDTA, 100 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate), high salt buffer (50 mM Tris-HCl, pH 8.1, 10 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate), LiCl2 buffer (10 mM Tris-HCl, pH 8.0, 0.25 M LiCl2, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA), and TE buffer. Bound chromatin was eluted and reverse-crosslinked at 65°C overnight. DNAs were purified using Min-Elute PCR purification kit (Qiagen, Germantown, MD) after the treatment of RNase A and proteinase K. The libraries were prepared from about 5 - 10 ng ChIP and input DNA using the ThruPLEX DNA-seq kit (TaKaRa, San Jose, CA). ChIP enrichment was validated by performing real-time PCR in library DNAs. The following primers were used: H3K27ac-positive control locus: SMARCA4-TSS-F: 5'-TTGGCGAAGCTGCGATCGGG-3', SMARCA4-TSS-R: 5'-AGGGGACCGCTAATGCCCGT-3'; ACTB-TSS-F: 5'-CCTCATGGCCTTGTCACAC-3', ACTB-TSS-R: 5'-GCCCTTTCTCACTGGTTCTCT-3'; H3K27ac-negative control locus: Ch19-intergenic-F: 5'-AGCTTGTCTTTCCCAAGTTTACTC-3', Ch19-intergenic-R: 5'-TAGCTGTCGCACTTCAGAGGA-3'.