ChIP-qPCR Analysis Protocol

ChIP analysis was performed as described previously [52 (link)]. Exponentially growing cells were fixed with 3% formaldehyde and then lysed in ChIP lysis buffer (140 mM NaCl, 50 mM HEPES-KOH pH 7.5, 1% Triton X-100, 1 mM PMSF, and 1% deoxycholate) with glass beads. DNA fragments were obtained by sonication, and anti-Flag M2 affinity agarose beads (Sigma, Beijing, China) were used for immunoprecipitation. DNA fragment-bound agarose beads were washed 3 times with ChIP lysis buffer (140 mM NaCl, 50 mM HEPES-KOH, pH 7.5, 1% deoxycholate, and 1% Triton X-100) and twice with wash buffer (10 mM Tris/HCl pH 7.5, 1 mM EDTA, 250 mM LiCl, 1% NP40, and 1% sodium deoxycholate). After crosslinking was reversed, immunoprecipitated DNA was digested with RNase A (Thermo) and Proteinase K (Thermo), followed by phenol/chloroform/isoamylol (25:24:1) extraction. DNA was precipitated by ethanol with 3 M sodium acetate and resuspended in TE buffer. The purified DNA was used for qPCR analysis. For ChIP quantification, we used the following formula as described previously [53 (link)]: enrichment = 2 ^{− ((Ci}_test^{− Ci}_act1^{)IP − (Ci}_test^{− Ci}_act1^)wce)), where Ci_act1 and Ci_test are the effective amplification cycles for the reference and test, respectively, in the input DNA (wce) samples and immunoprecipitated (IP) samples.