H3k56ac

In total, 10 ml of yeast culture was collected for each condition. Cell pellets were resuspended in 300 μl lysis buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 7 mM EDTA, 5 mM DTT, cOmplete mini protease inhibitor (Roche 4693124001)). The samples were then vortexed with glass beads (Biospec 11079105) at 4 °C for 20 min. Samples were centrifuged and the supernatant collected for analysis. Samples were loaded onto NuPAGE Novex 4 to 12% Bis-Tris protein gels (Invitrogen, NP0322). Gels were transferred to nitrocellulose membrane by wet transfer. Membranes were incubated with antibodies in PBS-0.2% tween with 5% milk. The following primary antibodies were used at the specified concentrations: H3 (CST 9715, 1:10,000), H3K4ac (Millipore 07-539, 1:1000), H3K9ac (Millipore 07352, 1:5000), H3K14ac (Millipore 07-353, 1:10,000), H3K18ac (Millipore 07-354, 1:5000), H3K23ac (Millipore 07-355, 1:10,000), H3K27ac (Millipore 07-360, 1:15,000), H3K56ac (Millipore 07-677, 1:1000), H3 pan-acetyl (Millipore 06-599, 1:500), H4 (Millipore 05-858, 1:1000), H4K5ac (Millipore 07-327, 1:2000), H4K8ac (Abcam ab15823, 1:1000), H4K12ac (Abcam ab1761, 1:5000), H4 pan-acetyl (Millipore 05-858, 1:1000), and pSTAIR loading control (Sigma-Aldrich P7962, 1:4000). Membranes were developed using ECL films (GE Healthcare Life Sciences, 28906836) and a XOGRAF Compact X4 film processor.

Cells (2 × 10⁶) were cross-linked with 1% paraformaldehyde for 10 minutes at room temperature and quenched by glycine. Cells were washed with cold PBS, centrifuged at 360g at 4°C for 5 minutes, and lysed. After sonication, samples were spun down and incubated with 1 μg primary antibody for each ChIP experiment at 4°C overnight. Magnetic beads (Thermo Fisher Scientific) were added the next day and incubated at 4°C for 2 hours. Samples were then washed, and histone complexes were eluted. The eluted samples were treated with RNase A and proteinase K, reversed crosslink, and purified with Qiagen PCR purification kit. The purified DNA samples were subjected to qPCR using the indicated gene-specific primers listed in Supplemental Table 2. Antibodies used for ChIP experiments are listed as follows: H3K4me1 (Abcam, ab8895), H3K4me3 (Active Motif, 39159), H3K27me3 (Cell Signaling Technology, 9733), H3K27ac (Abcam, ab4729), H3K36me3 (Abcam, ab9050), H3K56ac (Millipore, 07-677), and rabbit IgG (Abcam, ab171870). Data were normalized as percentage of input.