GSM4742959: I-BET151_ChIP_rep2; Homo sapiens; ChIP-Seq
Cells cultured in a 10-cm dish (about 10^7 cells) were collected and fixed in 1% formaldehyde at RT for 10 min. Then, 0.125 M glycine was used to quench the reaction. After washed with 1× PBS twice, cells were harvested and lysed by Lysis Buffer and Cell Lysis Buffer in Magna ChIP A/G One-Day Chromatin Immunoprecipitation Kit (EMD Millipore Corp, Cat. #17-10086) step by step. Thereafter, the nuclear pellets were resuspended in Nuclear Lysis Buffer in Magna ChIP A/G One-Day Chromatin Immunoprecipitation Kit. The mixture was sonicated as 30s on, 30s off with 20 cycles. The sonication samples were centrifuged at 12000 rpm for 20 min at 4°C to harvest supernatant. 20 μl Protein A/G magnetic beads (EMD Millipore Corp) for each immunoprecipitation were washed with block buffer (1× PBST and 0.5% BSA) for 2 times. Before immunoprecipitation, Protein A/G magnetic beads and 2 μg antibodies were incubated at 4°C overnight followed by washed using block solution for 3 times. Moreover, the sonication supernatant was diluted in dilution buffer to 1 ml total volume and 20 μl of the mixture was left as input samples. Next, the sonication supernatant was incubated with beads at 4°C for 6-10 hours. After incubation, the Protein A/G beads, antibodies and chromatin formed the complex. And the complex was stepwise washed in 1 ml wash buffer (20 mM Tris-HCl pH 8, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), high NaCl buffer (Wash supplemented with 500 mM NaCl) and TE buffer (10 mM Tris-HCl pH 8, 50 mM NaCl, 1 mM EDTA) at 4°C for 5min. Then, DNA fragments were eluted by incubating with 200 μl elution buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, 1% SDS) at 65°C for 30 min followed by reversed in elution buffer with 0.2 M NaCl at 65°C overnight. Finally, RNA in these samples was digested with 0.2 mg/ml RNase A at 37°C for 2 h and protein was digested with 0.2 mg/ml proteinase K at 62°C for 2h and 95°C for 10 min. DNA was extracted by using DNA Clean & Concentrator-5 Kit (ZYMO, Cat. D4014). ChIP and input DNA were quantified by Qubit before library preparation using the VAHTS Universal DNA Library Prep Kit for Illumina V2 (Vazyme).