GSM7814367: ChIP_IgG_siTAF5+TAF5-FL_Replicate2; Homo sapiens; ChIP-Seq
Cells were fixed with 1% formaldehyde (Pierce #PI28908) for 10 minutes and the fixation was stopped with 125 mM of glycine (Sigma #G8898) for 7 minutes. The cell pellets were resuspended in 45 mL of Farnham lysis buffer (5 mM PIPES pH 8.0_85 mM KCl and 0.5% NP-40) and incubated at 4°C for 20 minutes with gentle shaking. The supernatant was discarded by centrifugation_and the pellets were again resuspended in 25 mL of Farnham lysis buffer and incubated at 4°C for 15 minutes with gentle shaking. The purified nuclei pellets were subsequently resuspended in 0.8 mL of CHIP lysis buffer (50 mM HEPES-KOH pH7.5_140 mM NaCl_1 mM EDTA pH8.0_1% Triton X-100_0.1% Sodium Deoxycholate_0.1% SDS and protease inhibitors cocktail) and sonicated using a BioruptorPlus (Diagenode) at high power for 15 cycles (30 seconds on and 30 seconds off). The sheared chromatin was centrifuged at 18,500 rcf for 5 minutes_and the supernatant was collected. For each immunoprecipitation_50 μg of sheared chromatin_6 μg of the antibodies [rabbit IgG (Proteintech #30000-0-AP)_TBP (Proteintech #22006-1-AP)_and RNA polymerase II (ThermoFisher Scientific #A300653A)]_and 40 μL of Dyna-Protein G beads were used. The immunoprecipitations were performed at 4°C overnight using RIPA buffer. 10% of the sheared chromatin was kept aside as input. The beads were then washed twice with low salt (0.1% SDS_1% Triton X-100_2 mM EDTA_20 mM Tris-HCl pH 8.0 and 150 mM NaCl)_high salt (0.1% SDS_1% Triton X-100_2 mM EDTA_20 mM Tris-HCl pH 8.0 and 500 mM NaCl)_and LiCl wash buffers (0.25 M LiCl_1% NP-40_1% Sodium Deoxycholate_1 mM EDTA and 10 mM Tris-HCl pH 8.0). The chromatin was eluted using 100 μL of elution buffer (1% SDS and 100 mM NaHCO3) by incubating for 1 hour at room temperature and 15 minutes at 65°C. The eluted samples and input DNA were subjected to overnight decrosslinking followed by RNaseA (Invitrogen #12091021_0.2 mg/ml final concentration) and Proteinase-K (ThermoFisher Scientific #EO0492_0.4 mg/ml final concentration) treatment for 1 hour and purified using a QIAquick PCR Purification Kit (Qiagen #28104). The sequencing library was prepared from 11.5 ng of purified DNA using the Accel-NGS 2S Plus DNA Library Kit (Swift Biosciences # 21096)