title: GSM3656931: ChIP_Calibrated_Nz_Smc1-6HA Sth1-AID; Saccharomyces cerevisiae; [Candida] glabrata; ChIP-Seq
library_construction_protocol: Cells were fixed for 15 minutes at 25 °C, and quenched with glycine (final concentration 125 mM) for 7 minutes before cells were harvested by centrifugation at 4,000 r.p.m. for 1 minute. The cell pellets were washed in PBS and transferred to a screw cap tube and frozen on dry ice. The pellets were stored at -80 °C. Pellets were resuspended in 300 μl of IP buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, NP-40 (0.05% v/v), Triton® X-100 (1% v/v)) containing phenylmethanesulfonyl fluoride (PMSF, final concentration 1 mM) and Complete protease inhibitor cocktail (without EDTA, from Roche). 500 μl of glass beads were added to the tubes. Cells were broken in a FastPrep® FP120 (BIO101) by 3 repetitions of a 20 seconds cycle, power setting 5.5. Cells were maintained on ice for 2 minutes after each cycle. The tubes were pierced with a hot needle and placed into new eppendorf and spun at 2000 r.p.m. for 2 minutes to collect the lysate. 100 μl of IP buffer containing PMSF and protease inhibitors were then added and tubes spun again at 2000 r.p.m. for 1 minute. The cell lysate was spun down for 10 minutes at 15,000 r.p.m at 4 °C. This pellet was resuspended in 1ml of IP buffer containing PMSF and protease inhibitors, and sonicated for 1 hour (30 seconds on, 30 seconds off) at high power at 4 °C in a Diagenode Bioruptor. After sonication samples were spun down for 10 minutes at 15,000 r.p.m. and the supernatant taken. 200 μl of the sonicated chromatin were taken as “input” and 400 μl was incubated with 40 μg of HA antibody (anti-HA 12CA5 from Roche) in a sonicator at low power for 30 minutes (30 seconds on, 30 seconds off). The “input” DNA was precipitated with 0.3 M sodium acetate and 2.5 volumes of cold ethanol and spun down at 15,000 r.p.m. for 30 minutes, and the supernatant removed. The pellet was washed with 70 % ethanol and air dried. After antibody binding the IP sample was spun down at 13,000 r.p.m for 5 minutes and the supernatant added to 60 μl of Dynabeads protein G (Invitrogen) previously    equilibrated with IP buffer. Then the samples were incubated for 2 hours at 4 °C in a rotating wheel and washed 5 times with IP buffer using a magnetic separator rack. Finally “input” samples and IP samples were resuspended in de-crosslinking buffer (TE 1X, 1 % SDS, 10 μg/ml RNase A , 1 mg/ml proteinase K) and incubated at 65 °C overnight. Samples were purified using ChIP DNA Clean & Concentrator kit (Zymoresearch) according to the manufacturer instructions