p53 ChIP-seq Library Preparation

ChIP-seq library preparation was adapted from the Active Motif ChIP-IT High Sensitivity Kit protocol (Active Motif, 53040). A2780 cells were grown and treated in 150cm dishes until 80% confluency, aiming for 15 million cells per treatment prior to crosslinking. Cells were cross-linked for 10 minutes at room temperature. The samples were sheared using 2 μL MNase (NEB, M0247S) for 10 minutes, then briefly sonicated 4 cycles (30secs on/30secs off) at 4°C. Samples were then centrifuged and the supernatant was used as the input and for the downstream immunoprecipitation. The immunoprecipitation was performed following the manufacturer’s protocol of the Active Motif Kit, using 4 μL of the p53 antibody (Bethyl Laboratories, A300-247A-M). DNA was purified and used for qPCR analysis to verify enrichment prior to ChIP-Seq. ChIP-seq was completed using the NEBNext Ultra II DNA Library Prep (NEB, E7103S) with multiplex oligos for barcoding (NEB, E7335S).