Hsf1_ChIP
250 mL of mid-log growing yeast cells (OD595 = 0.6) were cross-linked with 2% formaldehyde for 30 min at 30oC, the reaction was quenched with glycine (final concentration = 125 mM), and cells were collected by centrifugation. Subsequently, cells were spheroplasted according to the protocol of the Rando lab (Rando, 2010) and then directly sonicated (Bioruptor, Diagenode: ten cycles, 30 sec on/off, medium setting. 200 µL chromatin extract was incubated with 10 µL of anti-GFP antiserum (3h, RT) which had been coupled to Protein G agarose beads (Roche 11 243 233 01) overnight at 4oC. After incubation antibody ChIPs were washed twice in FA lysis buffer (50 mM HEPES KOH pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS), twice with FA lysis buffer containing 0.5 M NaCl, and twice with 10 mM Tris at pH 8.0, 0.25 mM LiCl, 1 mM EDTA, 0.5% Nonidet P-40 and 0.5% Na-deoxycholate. Cross-links of the ChIP samples were reversed overnight at 65°C in 150 μL 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1% SDS. Samples were treated with RNAse and proteinase K, and DNA was recovered for further analysis using phenol extraction. Prior to library preparation the samples were sub-sheared to obtain fragments in the optimal size range (Mokry et al., 2010).