title: Rad2_HA_Rpb9
organism: Saccharomyces cerevisiae S288C
description: Protocols: The cell pellets are resuspended in 1 ml of FA/SDS/PMSF buffer (50 mM HEPES-KOH, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X100, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF) and transferred to 1.5 mL Eppendorf tubes. 0.75 mL glass beads (425-600 µm, Sigma) are then added to each tube, and the yeast cells were lysed at 4°C for 15 min by vortexing. Pierce the bottom of a 12 ml Greiner tube with a hot needle (0.7x30 mm or 0.5x15 mm) and place in a Falcon50 with a pierced cap. The lysates are transfered in the pierced tubes. Wash each Epp with twice 1 ml of FA/SDS/PMSF buffer (4 ml per extract). Spin down 1 min/2000 rpm. Gently resuspend the pellet with a 5 ml pipet and transfer in a sterile 15 ml Corex tube. Spin down 20 min / 12000 rpm / 4°C in a JA17 rotor (Beckmann). Vacuum the supernatant. The cross-linked chromatin appears as a transparent layer around the pellet. Transfer the pellet in a 2 ml Epp with a burned Pasteur pipet. Add 0.8 ml of FA/SDS/PMSF buffer in the Corex and resuspend with the Pasteur pipet. Transfer in the Epp. Wash the Corex with 0.8 ml FA/SDS/PMSF buffer. Homogenize the Epp with the Pasteur pipet and incubate on a rotating wheal at 4°C for 1 to 2 hours.Spin down 20 min / 12000 rpm / 4°C. Vacuum the supernatant and resuspend in 1.6 ml FA/SDS/PMSF buffer with a burned Pasteur pipet. Sonication step was performed on a S220 focused-ultrasonicator (Covaris) in 1mL milliTube (Covaris), for two cycles of 3 minutes spaced by a 30s rest time. Each cycle consisted of 150W pulses for 10% of the time (duty factor 10). Sonicated lysates were then transferred to a new 2mL safe-lock tube and centrifugation was performed at 15000g for 20 minutes. Supernatant was collected and combined with 300µL FA/SDS/PMSF. Sonicated chromatin was divided in 250µL aliquots, snap-frozen in liquid nitrogen and kept at -80°C until further use. Immunoprecipitation, DNA precipitation and library preparation were done on an IP-Star compact automated system (Diagenode) using built-in programs and following manufacturer's instructions, with the following exceptions (Denby Wilkes et al., in preparation). IP was done using the ChIP_IPure_200_D program, at 22°C, with 1h “Ab coating” (Slow speed), 3h “IP reaction” (Medium speed) and 5 minutes “washes” (Fast speed). “Beads wash buffer” was PBS + 0.1% BSA, FA/SDS adjusted to 500mM NaCl was used for “IP Wash 1” and “IP Wash 2”, “IP Wash 3” was done with IP Buffer (Tris 10mM pH8, LiCl 0.25M, EDTA 1mM, NP40 0.5%, Na-Deoxycholate 0.5%), “IP Wash 4” was done with TE (Tris 10mM pH 8, EDTA 1mM). “Elution buffer” was the elution buffer (A+B) of Auto iPure kit V2 (Diagenode). “Ab coating mix” was 1µL anti-HA (12CA5), 2µL anti-Myc (9E10) or 5µL anti-Rpb1-CTD (8WG16) completed to 100µL with “Beads wash buffer”. “Sample” was 200µL sheared chromatin, supplemented with 4µL BSA and 4µL 50X Protease Inhibitor Cocktail (prepared by dissolving one cOmplete tablet in 1mL ddH2O). 20µL DiaMag protein A-coated magnetic beads (Diagenode) were used per sample. After program completion, strips containing the eluates were warmed to redissolve precipitated SDS, eluates were recovered using a magnetic stand, 5µL of 5M NaCl was added to eluates and they were incubated for 4h at 65°C to reverse crosslink. 1µL RNase A (ThermoFisher) was added to eluates and they were then incubated for 30 minutes at 37°C. After reaching exponential phase at 30°C in YPD, cells were cross-linked with 1% formaldehyde during 10 minutes. DNA was extracted using the Auto iPure V2 kit from Diagenode, following the manufacturer recommendations. Library construction was done according to the MicroPlex Library Preparation Kit v2 (Diagenode) protocole, following manufacturer's recommandation. Libraries were sized with AMPure beads (Beckman-coulter) to a final size of 400bp.