Spot 4.1.1 imaging software

Colony color assays were performed as described previously [72 (link),73 (link)]. Logarithmically growing yeast that contain ADE2 between the E and I silencers at HMR were plated on rich (YPD) media containing varying concentrations of DHC and/or MA. Plates were incubated at 30°C for 2 days and stored at 4°C for three days to develop the color. Images were taken using a Leica MZ125 microscope and SPOT 4.1.1 imaging software [72 (link)]. In this assay, red colonies indicate that ADE2 at HMR is silenced, white colonies indicate that ADE2 is expressed, pink colonies indicate a defect in maintaining or inheriting silencing of ADE2 (defect in the stability of silent chromatin), and sectored colonies signify a defect in establishing silencing (defect in forming silent chromatin de novo). In these experiments, media was autoclaved, cooled to 50–55°C in a water bath, then DHC, MA or DHC plus MA added to media just prior to pouring plates. Plates were used within ~24 hr.

Ten-fold serial dilutions of yeast containing TRP1 integrated between the E and I silencers at HMR were plated onto minimal (YM) media lacking tryptophan or synthetic complete media containing varying concentrations of DHC and incubated for two days at 30°C. Images were taken using a Leica MZ125 microscope and SPOT 4.1.1 imaging software. In these experiments, media was autoclaved, cooled to 50–55°C in a water bath, then DHC was added to media just prior to pouring plates. Plates were used within ~24 hr.