GSM5217305: dDSB Spo11-myc18 Rec104-FRB-HA3 rad50S t4.5 + Rapamycin t4 WCE; Saccharomyces cerevisiae; Saccharomyces kudriavzevii; OTHER
At the desired time points, cells were harvested from 100 ml meiotic cultures and collected by centrifugation (4 min, 2,000 rpm , 4°C), washed once with ice-cold 1xTBS, centrifuged again and pellets were frozen at -80°C. Cells pellets were lysed in 2.4 ml ice-cold lysis buffer (50 mM Hepes-KOH pH=7.5, 150 mM NaCl, 5 mM EDTA, 1% Trition X-100, 0.1% Na-deoxycholate, 1 protease inhibitor tablet (Roche, Complete Protease Inhibitor Cocktail)). For spike-in experiments, 20-30 % of S. kud. meiotic cells were added to S. cer. cells. All steps were carried out at 4°C unless stated. Cells were opened with glass beads 2 x 20 sec, 5.5 m/s in a FastPrep-24™ 5G Instrument (MP Biomedicals). The cell lysates were centrifuged at full speed for 10 min at 4°C and the supernatants were transferred into new Eppendorf tubes. A pre-clearing step was performed by incubating the soluble chromatin fractions with Dynabeads Pan Mouse IgG magnetic beads (Invitrogen) for 1 hour at 4°C. After removing 40 µl of the lysate for the whole cell extract (WCE), chromatin immunoprecipitation was carried out by incubating the lysates with Dynabeads Pan Mouse IgG magnetic beads (Invitrogen) coupled to anti-myc antibody (9E11, mouse monoclonal AB) for 3 hours at 4°C. Thereafter, the beads were washed 2 times with 1 ml lysis buffer, 2 times with 1 ml lysis buffer high salt, 3 times with 1 ml washing buffer (10 mM Tris-HCL pH=8.0, 250 mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate, 5 mM EDTA) and finally 2 times with 1 ml 10 mM Tris-HCl pH=8.0. Unprotected DNA or partially protected DNA was digested with 3 µl ExonucleaseV (NEB) in NebBuffer 4 (50 mM K-acetate, 20 mM Tris-acetate, 10 mM Mg-acetate and 1 mM DTT at pH=7.9 containing 1 mM ATP) for 30 min at 37°C. In parallel, RNA was removed by incubation with 10 µl RNase (Dnase-free, Roche). The reaction was stopped by the addition of 11 mM EDTA, the beads were washed 3 times with ice-cold 1xTE and incubated at 65°C for 30min in elution buffer (50 mM Tris-HCL pH=8.0, 10 mM EDTA, 1 % SDS). Spo11-protected DNA complexes were digested over night using 12 µl Proteinase K (Sigma) at 50°C. DNA was extracted once with phenol/chloroform/isoamyl alcohol (25:24:1) and precipitated with 0.5 μg/μl glycogen (Roche), 200 mM NaCl and 2.5 volumes of 96 % ethanol. The DNA was pelleted by centrifugation at 4°C for 1 hour, washed once with 1 ml of 70% ethanol and centrifuged again. After air-drying the pellet for 5 min, the DNA was resuspended in 60 µl 10 mM Tris-Cl pH=8.0. The WCE control was digested with RNase and proteinase K, followed by DNA extraction exactly as with the immunoprecipitated samples. The immunoprecipitated DNA was digested with 3 µl hTdp2 (BPS Bioscience) in 1x hTdp2 Buffer (50 mM Tris-HCl pH7.4, 1 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.1 mg/ml BSA) at 25°C for 30 min. The DNA was extracted with phenol/chloroform/isoamyl alcohol (25:24:1) and precipitated as described above. The hTdp2 digested DNA was end-repaired, A-tailed and adapters were ligated to the DNA ends using the NEBNext Ultra II DNA Library Preparation Kit (NEB) according to the manufacturer´s instructions except that we aimed to select for fragments between 140 bp  - 1000 bp length using Agencourt AMPure XP beads (Beckman Coulter). This necessary step likely results in under-representation of the shortest inserts. For amplification, we used either the NEBNext multiplex oligos or the NEBNext dual indexes primer sets. Before library preparation of the WCE control, we fragmented the DNA using NEBNext dsDNA fragmentase (NEB) according to the manufacturer´s protocol. Briefly, we incubated 32µl of the WCE control in 1x fragmentase reaction buffer V2 (50 mM NaCl, 20 mM Tris-HCl, 0.1 mg/ml BSA, 0.15% Triton® X-100, 15 mM MgCl2, pH 7.5@25°C) with 4µl NEBNext dsDNA fragmentase at 37°C for 20 min. The reaction was stopped by the addition of 5 µl of 0.5 M EDTA and the DNA was extracted by phenol/chloroform/isoamyl alcohol (25:24:1) and precipitated as described above. The library preparation of the WCE control was performed the same way as for the immunoprecipitated samples. The concentration and the molarity of the library were determined by NanoDrop™ 3300 Fluorospectrometer and Agilent 2100 Bioanalyzer.