GSM1568193: Cnp1_HA_10min_XIN_(20140725_6); Schizosaccharomyces pombe; ChIP-Seq
ChIP assays were performed using a protocol described previously (PMID: 22536162) with slight modifications. Briefly an exponentially growing culture of S. pombe strain was fixed with 1% formaldehyde for 15 min and the reaction was quenched for 5 min at room temperature using glycine to a final concentration of 125 mM. Cells were resuspended in resuspension buffer (0.2 mM Tris-HCl pH, 9.4, 10 mM DTT) and incubated at 37degC for 15 min on a shaker at 180 rpm. Spheroplasting (95%) was performed using Zymolyase-100T (amsbio, Cat # 120493-1) in spheroplasting buffer (1.2 M Sorbitol, 20 mM Na-HEPES, pH 7.5) at 37degC at low speed (80 rpm). Spheroplasting was terminated by adding ice-cold post-spheroplasting buffer (1.2 M Sorbitol, 1mM Mgcl2, 20 mM Na-PIPES, pH 6.8). Spheroplasts were pelleted at low speed and subsequently washed with ice-cold 1xPBS, Buffer I (0.25% TritonX-100, 10 mM EDTA, 0.5 mM EGTA, 10 mM Na-HEPES, pH 6.5), Buffer II (200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Na-HEPES). Washed spheroplasts were resuspended in extraction buffer (140 mM NaCl, 1 mM EDTA, 50 mM K-HEPES, 0.1% sodium deoxycholate, 1% Triton X-100, pH 7.5) with protease inhibitor cocktail to release chromatin. Released chromatin was digested with MNase in the presence of 3mM CaCl2 at 37degC for 10 min. Next, Chromatin was further sheared using bioruptor and the soluble fraction was obtained by centrifuging the chromatin solution at 13000 rpm for 15 min at 4degC. To about 1/20 of total soluble chromatin equal volume of elution buffer I (50 mM Tris-HCl, pH 8, 10 mM EDTA, 1% SDS) was added and incubated at 65degC overnight to reverse crosslinking. Decrosslinked solution was treated with RNase A (Roche # 1119915) at 37degC for 30 min and Proteinase K (Roche Cat # 1 000 144) at 55degC for 1 hr. The DNA was extracted with an equal volume of phenol/chloroform/isoamyl alcohol and precipitated with ethanol. The DNA pellet was resuspended in TE.