GSM7720713: LNCaP_KRAB_H3K27ac_18h_rep1; Homo sapiens; ChIP-Seq
For each replicate, 20 million cells were fixed with 1% formaldehyde for 10 minutes at 24°C, quenched with 0.125 M glycine for 5 minutes, washed 2x with PBS, and stored at -80°C until use. The fixed pellets were lysed in 1% SDS lysis buffer and sonicated for 30 cycles (30s on/off) in a temperature-controlled Bioruptor 3000 to obtain a size range of 250-400 bp. After centrifugation at 15000 RPM for 20 minutes to remove debris, individual samples were incubated with Protein A/antibody-conjugated beads overnight while rocking at 4°C. The samples were washed six times with increasing salt buffers and DNA was eluted at 65°C overnight. All samples were treated with RNase A for 30 minutes at 37°C followed by Proteinase K at 65°C for 1 hour. DNA was purified using phenol chloroform. ChIP sequencing libraries were constructed using the KAPA Hyper Prep Kit (Roche; cat. 08278539001) following the manufacturer's instructions, using 20 ng DNA per sample. The libraries were assessed for quality, purity, and size using DNA High Sensitivity Bioanalyzer chips (Agilent; cat. 5067-4626), and those passing quality control (with an equal size distribution between 250-400 bp, no adapter contamination peaks, and no degradation peaks) were quantified using the Library Quantification Kit (Roche; cat. 07960298001). Finally, the libraries were pooled to a final concentration of 10nM and sequenced at the WCM Genomics Core on a NovaSeq 6000. The ChIP-seq utilized the ERalpha (ERa) antibody from Santa Cruz Biotechnology (Cat# sc-8002, AB_627558) to generate MACC peaks, along with the H3K27ac antibody from Abcam (Cat# ab4729, AB_2118291).