GSM8289675: NK92_HS_HSF1_IP_2; Homo sapiens; ChIP-Seq
Cells were washed in 1XPBS and fixed with 1% formaldehyde for 10 min at room temperature. Subsequent steps until elution used 1x protease inhibitor. Nuclei were isolated on ice using nuclei isolation buffer for 5 minutes, with additional 5 min incubation with NP-40 0.3%. The cell pellet was lysed on ice using nuclei lysis buffer. Samples were sonicated using Covaris sonication. Clarified samples were transferred to Eppendorf tubes and diluted 1:10 with IP dilution buffer. Samples were precleared with 25 µl Protein A by rotation at 4°C for 1h. Supernatants were transferred to fresh tubes, and antibody was added and incubated overnight at 4°C. The next day, 25 µl of Protein A Dynabeads were added and samples were rotated at 4°C for 3 hours. After bead captures, beads were washed twice sequentially in each of. The following buffers: low salt wash buffer, high salt wash buffer, LiCl wash buffer, and TE. Samples were eluted and treated with RNase at 37°C for 30 minutes. Crosslinks were reversed overnight with the addition of proteinase K 0.5 mg/ml and 0.5% SDS. DNA was isolated using phenol/chloroform extraction followed by precipitation with ethanol and glycogen. Libraries were prepared using the Swift biosciences DNA low-input library kit