Uno stage top incubator

Live-cell imaging of transcription dynamics was performed as previously described in detail with minor modifications (40–42 (link)). In brief, yeast cultures were started in synthetic complete medium containing 2% raffinose in the morning, diluted in the evening and grown O/N to mid-log (OD_600 nm 0.2–0.4) whereafter they were imaged on a coverslip with a 2% agarose pad containing 2% galactose and 2% raffinose. Imaging was performed on a setup consisting of an AxioObserver inverted microscope (Zeiss), an alpha Plan-Apochromat 100× NA 1.46 oil objective, an sCMOS ORCA Flash 4v3 (Hamamatsu) with a 475–570 nm dichroic (Chroma), 570 nm longpass beamsplitter (Chroma) and 515/30 nm emission filter (Semrock), a UNO Top stage incubator (OKOlab) at 30°C, and LED excitation at 470/24 nm (SpectraX, Lumencor) at 20% power and an ND 2.0 filter, resulting in a 62 mW/cm² excitation intensity. Widefield images were recorded for 1 h at 15 s interval, with z-stacks (9 planes at 0.5 μm intervals) and 150 ms exposure using Micro-Manager software (43 (link)). For each condition, 9 replicate datasets were acquired with in total at least 265 cells.

Live-cell imaging of transcription dynamics was performed as previously described in detail^{4 (link),57 (link)} with minor modifications. In brief, cells were treated with 7.5 μM rapamycin or dimethyl sulfoxide (DMSO) for 60 min and subsequently imaged at mid-log (optical density (OD_600nm) 0.2–0.4) on a coverslip with an agarose pad consisting of 2% agarose and synthetic complete medium containing 2% galactose and 7.5 mM DMSO or rapamycin. Imaging was performed on a setup consisting of an AxioObserver inverted microscope (Zeiss), an alpha Plan-Apochromat ×100 numerical aperture (NA) 1.46 oil objective, an sCMOS ORCA Flash 4v.3 (Hamamatsu) with a 475–570 nm dichroic (Chroma), 570 nm longpass beamsplitter (Chroma) and 515/30 nm emission filter (Semrock), a UNO Top stage incubator (OKOlab) at 30 °C and light emitting diode (LED) excitation at 470/24 nm (Spectra X, Lumencor) at 20% power and an neutral density (ND) 2.0 filter, resulting in a 62 mW cm⁻² excitation intensity. Widefield images were recorded for 1 h at 15 s intervals, with z-stacks (nine slices, Δz 0.5 μm) and 150 ms exposure using Micro-Manager software^{58 (link)}. For each condition, at least two and often three replicate datasets were acquired with at least 80 cells in total.