GSM8486544: shA4,PLKO,PolII,ChIP; Homo sapiens; ChIP-Seq
Cells were fixed in complete media with 0.3% formaldehyde for 30 minutes at 4°C, then quenched by adding 0.125 M glycine for 5 minutes at room temperature and 15 minutes on ice. Fixed cells were pelleted and washed once with 1× PBS before snap-freezing on dry ice. cell pellet was resuspended in 1 ml buffer A (25 mM HEPES-NaOH, pH 7.5; 10 mM KCl; 0.1% NP-40; 1.5 mM MgCl2) and homogenized. Nine volumes of buffer B (15 mM HEPES-NaOH, pH 7.9; 15 mM NaCl; 60 mM KCl; 0.5 mM PMSF; 0.32 M sucrose) were added, and the mixture was centrifuged at 2000 RPM (4°C, 7 minutes) to collect nuclei. The nuclei were resuspended in 1 ml buffer B and 3.3 µl of 1 M CaCl2, then prewarmed at 37°C for 15 minutes. MNase (1 µl, NEB, M0247SXXX) was added and incubated at 37°C for 15 minutes. The reaction was quenched with 10 µM EGTA on ice for 5 minutes. One volume of 2x buffer C (20 mM Tris-HCl, pH 8.0; 200 mM NaCl; 2 mM EDTA; 1 mM EGTA; 0.2% Na-Deoxycholate; 1% N-lauroylsarcosine) and Triton X-100 (final 1%) were added. Samples were then passed through a 21G needle five times and centrifuged at 13000g (4°C) for 15 minutes. Five micrograms of antibodies were added to the lysate for overnight incubation at 4°C. Protein G Magnetic Dynabeads® (ThermoFisher Scientific) were used for pulldown. Immunoprecipitated chromatin bound to Dynabeads was washed four times with RIPA buffer (50 mM HEPES-KOH, pH 7.5; 500 mM LiCl; 1 mM EDTA; 1% NP-40; 0.7% Na-Deoxycholate). Chromatin was eluted from Dynabeads in 100 µl elution buffer (50 mM Tris-HCl, pH 8.0; 10 mM EDTA; 1% SDS) by incubating at 65°C for 30 minutes. Immunoprecipitated samples and input were incubated overnight at 65°C to denature formaldehyde crosslinking. Samples were then treated with RNase A (ThermoFisher Scientific) followed by proteinase K (Sigma Aldrich) before extraction using a Zymo kit. ChIP-sequencing (ChIP-seq) libraries were constructed using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer's instructions.