p300 Chromatin Immuno-precipitation (ChIP) assay followed by Next-gen sequencing (ChIP-Seq) and analysis

PC3 cells were treated with or without 30μm MnTE-2-PyP overnight, and then irradiated with 20 Gy. One hour post-irradiation, cells were fixed, lysed (Active Motif ChIP-IT Express #53008) and sheared for Ion Proton ChIP-seq. DNA-protein complexes were immunoprecipitated with an anti-p300 ChIP-grade antibody (Abcam #54984), and the genomic DNA purified (Invitrogen Magnify ChIP Kit #49-2024). The isolated genomic DNA was processed for next-gen library construction using the Ion Plus Fragment Library Kit #4471252. Briefly, libraries were constructed and analyzed for size and quantity using the High Sensitivity Bioanalyzer Kit (Agilent Technologies #5067-4626). The libraries were then sequenced with the Life Technologies Ion Proton, at the NJH Genomics Facility. Total input control libraries were used to normalize ChIP-seq signals, enabling peak comparison in the ChIPed samples. ChIP-sequencing reads were aligned to the human reference genome (hg109) using Ion Torrent Suite software. Peak-calling from aligned reads was performed using the Model-based Analysis for ChIP-Sequencing (MACS2) software [12], comparing p300 IPs to their respective total input controls. Peak significance was determined with a false discovery rate cutoff of 0.001. Peaks found by ChIP-seq were associated with the nearest gene by genomic location, using both the RefSeq, refGene, and UCSC known gene models. The gene list was narrowed by sorting for genes with peak Q-value greater than 10. Gene lists were converted to Ensembl annotation using Biomart and analyzed with GATHER Duke Bioinformatics system [13]. All data is representative of at least two biologically independent ChIP experiments.