GSM9487021: H2A-P_H2AS129A_Log_K_Rep1 (Input); Saccharomyces cerevisiae; ChIP-Seq
4 × 10⁸ S. cerevisiae cells were mixed with ~1 × 10⁸ Schizosaccharomyces pombe cells expressing Rad21–6HA, fixed in 1% formaldehyde for 15 min at room temperature (RT), and quenched with 150 mM glycine for 5 min. Cells were pelleted, washed once with TBS, and resuspended in 600 µl FA lysis buffer (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 0.1% sodium deoxycholate, 1% Triton X-100, protease inhibitor cocktail, 1 mM PMSF). Cells were lysed by four cycles of bead beating (6,500 rpm, 30 s) and lysates were sonicated to an average DNA fragment length of ~600 bp. After centrifugation for 30 min at 15,000 rpm, cleared supernatants were transferred to new tubes and 1% of each was kept as an input sample. For immunoprecipitation, 5 µl of antibody against the S. cerevisiae protein of interest and 2 µl of anti-HA antibody recognizing Rad21–6HA were added to the lysates, and samples were incubated overnight at 4°C with rotation. The next day, DNA-protein complex es were captured with Protein G Dynabeads and sequentially washed with FA-lysis buffer, FA-500 buffer (50 mM HEPES–KOH pH 7.5, 500 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM EDTA), LiCl wash buffer (10 mM Tris–HCl pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA) and TE (10 mM Tris–HCl pH 7.5, 1 mM EDTA) on a rotator for 5 min each time. Beads and input samples were then resuspended in elution buffer (50 mM Tris–HCl pH 7.5, 1% SDS, 10 mM EDTA) and incubated O/N at 65°C on a thermal shaker. The next day, magnetic beads were removed, and all samples were treated with RNase A (100 µg/ml) for 1 h at 37°C, followed by a 1 h incubation at 65°C in the presence of proteinase K (1 mg/ml). DNA from H2A-S129-P, Rap1-13cMyc, Ku70-13cMyc, Tel1-12Pk, Pol2-3HA, and Rpb1 ChIP samples was purified using a ChIP DNA Clean & Concentrator kit (Zymo Research). DNA from RPA, Rad52–YFP, Rad51, Rad59–13cMyc, and Cdc13–18cMyc ChIP samples was extracted by phenol–chloroform purification and ethanol precipitation. Sequencing libraries for H2A-S129-P, Rap1-13cMyc, Ku70-13cMyc, Tel1-12Pk, Pol2-3HA, and Rpb1 ChIP samples were prepared using the Novogene NGS DNA Library Prep Set. Libraries for RPA, Rad52–YFP, Rad51, Rad59–13cMyc, and Cdc13–18cMyc ChIP samples were generated using the xGen™ ssDNA & Low-Input DNA Library Preparation Kit (Integrated DNA Technologies) according to the manufacturer's instructions.