Cells were grown in YE medium and chromatin isolation and immunoprecipitation were carried out as previously described (Sanso et al., 2011 (link)), with minor modifications. Briefly, cells from 50-mL cultures were cross-linked with 1% formaldehyde for 10 (Clr3-Myc), 15 (Atf1-HA and H3K4me3) or 20 min (H3K9me2 and H3K9ac). Crosslinking was stopped with 125 mM glycine and after lysis of pellets with a bead beater, the lysates were sonicated in order to obtain chromatin fragments of ∼400 bp average size. Once the chromatin was isolated, it was immuno-precipitated with specific antibodies [5 μL of anti-HA antiserum (12CA5; house-made), 1 μL of anti-Myc (Merck Life Science, C3956), 1 μL of anti-H3K9me2 (Abcam, Ab1220) or 1 μL of anti-H3K9ac (Millipore) overnight at 4 °C rotating. Beads were washed, DNA was eluted and formaldehyde cross-linking was reversed. After protein digestion and chromatin extraction, DNA was amplified by quantitative PCR using Light Cycler 480 SYBR Green I Master (Roche). The error bars (SD) were calculated from at least three biological replicates, unless indicated otherwise. Primers from a mitochondrial DNA region or from act1 ORF gene was used as a negative control, as indicated, and they are listed in Table S2 .
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