GSM9724231: PC-3 WT, RNA Pol II ChIP, Replicate 3; Homo sapiens; ChIP-Seq
Wild-type (WT) and PSD10-knockout (SLK-KO) PC-3 cells were grown to 80% confluence. 40 million cells were cross-linked in 22 mL complete media with 37% formaldehyde to a final concentration of 1% for 8 minutes at 37°C with intermittent agitation. Cross-linking was quenched by addition of 2.5 M glycine (final 125 mM) for 5 minutes at room temperature. Cells were pelleted at 500 × g for 5 minutes at 4°C, washed twice with PBS supplemented with protease inhibitors, and aliquoted at 10 million cells per aliquot in Bioruptor TPX tubes. Pellets were lysed with 300 μL ice-cold lysis buffer (50 mM Tris-HCl pH 7.4, 1% SDS, 0.25% sodium deoxycholate) for 10 minutes on ice. Chromatin was fragmented by sonication using a Bioruptor (high power, 4°C, 40 cycles of 30 s on/30 s off) to target fragment sizes of 200-1000 bp. Lysates were diluted 4-fold with ChIP dilution buffer (50 mM Tris-HCl pH 7.4, 0.1% SDS, 150 mM NaCl, 1.84% Triton X-100) and clarified at 17,000 × g for 10 min at 4°C. For immunoprecipitation, 1.5 mL lysate (~5 million cells) was incubated with 4 μg antibody overnight at 4°C. Antibodies used: anti-MYC (Abcam AB32072), anti-POL2 (Abcam AB26721), anti-IgG (Cell Signaling 2729S). Antibody-chromatin complexes were captured with Protein G Dynabeads (1 mg/sample, 2 h, 4°C). Beads were washed sequentially with RIPA 150, RIPA 500, LiCl, and Tris-HCl buffers. Complexes were eluted in 100 μL elution buffer (10 mM Tris-HCl pH 8, 0.1% SDS, 150 mM NaCl, 5 mM DTT) at 65°C for 1 h. RNA was digested with RNase A (37°C, 30 min); crosslinks reversed with Proteinase K (65°C, overnight). DNA was purified using AMPure XP beads (1.8× volume) and eluted in 40 μL molecular grade water. Sequencing libraries were prepared using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (E7645S) from purified ChIP DNA following the manufacturer's protocol. Quality control and sequencing library preparation were performed by the Herbert Wertheim UF Scripps Genomics Core Facility.