GSM7099704: 7209_input (biol rep 2 mutant); Saccharomyces cerevisiae; ChIP-Seq
The 200 OD600 units harvested for each strain were split into four Eppendorf tubes (50 OD600 units in each) and resuspended in 400 µl of bead-beating buffer (50 mM HEPES-KOH pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 cOmplete Mini, EDTA-free Protease Inhibitor Cocktail tablet (Roche, 11836170001) per 5 ml of buffer). An equal volume of glass beads (0.5 mm diameter, BioSpec Products) was added, and the tubes were vortexed for 25 cycles (3,000 rpm; 30 seconds of vortexing, 1 minute of resting on ice). After vortexing, cells were separated from the beads and cells and nuclei were lysed with 0.5% (w/v) SDS for 10 minutes on ice. Tubes were then centrifuged at 20,817 g for 15 minutes at 4°C, the resulting supernatants were removed, the pellets were washed with 1 ml of 0.1% SDS buffer (50 mM HEPES-KOH pH 7.5, 150 mM NaCl, 0.1% (w/v) SDS, 1% Triton X-100 (w/v), 0.1% Sodium Deoxycholate, 1 mM EDTA, 1 mM PMSF, 1 cOmplete Mini, EDTA-free Protease Inhibitor Cocktail tablet (Roche, 11836170001) per 5 ml of buffer) and resuspended in 300 µl of the same buffer. Cell extracts were then sonicated in Bioruptor Plus sonicator (Diagenode) for 20 cycles (30 seconds ON / 30 seconds OFF) on the HIGH power setting. After the sonication, cell debris was spun down at 20,817 g for 15 minutes at 4°C, and supernatants were transferred into fresh Eppendorf tubes containing 1 ml of the 0.1% SDS buffer. Samples belonging to the same strain (split into 4 Eppendorf tubes at the beginning) were then pooled together. 10 µl were collected as input aliquots for each sample, mixed with 390 µl of TE (10 mM Tris-HCl, pH 8, 1 mM EDTA) and stored at -80°C. Four immunoprecipitation reactions were set up for each sample by mixing 1 ml of sonicated cell lysate with 15 µl of Anti-Rad51 (y-180) rabbit polyclonal (Santa Cruz Biotechnology sc-33626) antibody and 15 µl of Dynabeads protein A magnetic beads (Invitrogen, 10002D). The mixtures were incubated overnight at 4°C on a rotating wheel. Next day, beads were recovered using a magnet and sequentially washed with wash buffer 1 (50 mM HEPES-KOH pH 7.5, 275 mM NaCl, 0.1% (w/v) SDS, 1% Triton X-100 (w/v), 0.1% Sodium Deoxycholate, 1 mM EDTA, 1 mM PMSF, 1 cOmplete Mini, EDTA-free Protease Inhibitor Cocktail tablet (Roche, 11836170001) per 5 ml of buffer), wash buffer 2 (50 mM HEPES-KOH pH 7.5, 500 mM NaCl, 0.1% (w/v) SDS, 1% Triton X-100 (w/v), 0.1% Sodium Deoxycholate, 1 mM EDTA, 1 mM PMSF, 1 cOmplete Mini, EDTA-free Protease Inhibitor Cocktail tablet (Roche, 11836170001) per 5 ml of buffer), wash buffer 3 (10 mM Tris-HCl, pH 8, 0.25 M LiCl, 1mM EDTA, 0.5% IGEPAL, 0.5% Sodium Deoxycholate) and wash buffer 4 (10 mM Tris-HCl, pH 8, 1 mM EDTA) for 5 minutes each wash on a rotating wheel at room temperature. To elute precipitated chromatin, beads belonging to the same sample were pooled together in 200 µl of TES (50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1% SDS) and then incubated at 65°C for 10 minutes with mixing. Tubes were then vortexed, incubated at 65°C for additional 10 minutes and the eluted chromatin was separated from the magnetic beads by centrifugation at 15,871 g for 3 minutes. The beads were washed with 200 µl of TE on the rotating wheel for 15 minutes at room temperature. The tubes were then centrifuged at 15,871 g for 3 minutes, and the resulting supernatants were mixed with the corresponding aliquots of the eluted chromatin.  To remove proteins from the IPed DNA, Proteinase K was added to the immunoprecipitated chromatin samples to the final concentration of 1 mg/ml, and the tubes were incubated at 65°C overnight. Next day, immunoprecipitated DNA was purified using QIAquick® PCR Purification Kit (Qiagen, 28106) and eluted in 35 µl of water. ChIP-seq libraries were prepared using DNA SMARTTM ChIP-Seq Kit (Takara Bio, 634865) according to the manufacturer's instructions 12 samples were indexed as indicated in the sample description. Indexed samples were pooled and sequences together.