GSM8253840: CDK7_DMSO_Serum0; Homo sapiens; ChIP-Seq
For each ChIP, 1 x 15-cm tissue culture dish (yielding ~30E6 cells/plate) was utilized and seeded according to the experimental design in each figure (DMSO/dTAG +/- serum at different time points), and 20E6 cells were utilized per ChIP. For all Pol II ChIPs, cells were crosslinked with 0.5% methanol-free formaldehyde (Thermo Fisher Scientific, 28908) by adding directly to the dish in media at room temperature for 10 min with rocking and neutralization with 150 mM glycine for 5 min with rocking. For the ChIPs including HA (KAP1), SPT5, CDK9, TFIIB, MED1, and CDK7, 1% formaldehyde for 10 min was used instead of 0.5%. For all experiments, the media was removed and cells were washed twice with cold 1X PBS, cold PBS was added and then cells were scraped off the plate. Cells were centrifuged (1000xg, 5 min, 4°C), and pelleted, flash frozen in liquid nitrogen, and frozen at -80°C until ready to use. To perform the ChIP, cells were resuspended in 2 mL/dish of Farnham Lysis Buffer (5 mM PIPES pH = 8.0, 85 mM KCl, 0.5% NP-40, 1 mM PMSF, 1X Protease Inhibitor (RPI, catalog P50900-1)), counted by hemacytometer, resuspended to 10E6 cells/mL, nutated for 30 min at 4°C, and then centrifuged to isolate nuclei (1000xg, 5 min, 4°C). The supernatant was removed and nuclei resuspended in Szak's RIPA Buffer (50 mM Tris-HCl pH = 8.0, 1% NP-40, 150 mM NaCl, 0.5% Na-Deoxycholate, 0.1% SDS, 2.5 mM EDTA pH = 8.0, 1 mM PMSF, and 1X Protease Inhibitor) at a concentration of 25E6 nuclei/mL. Libraries were prepared using the KAPA Hyper Prep Kit (KAPA Biosystems, catalog KK8502) following the manufacturers protocol.