Yeast RNA Extraction and Reporter Assay

Cell cultures were inoculated from stationary phase saturated cultures grown overnight in -Leu DO media. The cultures were then grown until OD₆₀₀ = 0.6 −0.8, and 10 OD₆₀₀ units were collected by centrifugation. Total RNA was isolated from yeast and contaminating DNA was depleted using the MasterPure Yeast RNA Purification Kit (Epicentre, Madison, WI) protocol with minor changes as previously described (Carrocci et al., 2017 (link)). IR700 dye conjugated probes (Integrated DNA Technologies, Skokie, IL) were used for primer extension of the ACT1-CUP1 reporter (10 pmol yAC6: /5IRD700/GGCACTCATGACCTTC) and U6 snRNA (2 pmol yU6: /5IRD700/GAACTGCTGATCATGTCTG) (Carrocci et al., 2017 (link); van der Feltz et al., 2021 (link)). Primer extension products were visualized on a 7% (w/v) denaturing polyacrylamide gel (42 cm × 22 cm × 0.75 mm) run at 35W for 80 min at RT. Gels were imaged with an Amersham Typhoon NIR laser scanner (Cytiva), and band intensities were quantified with Image J (version 1.53v, 2022).