Glass bottom 1.5h n 96 well plates

For microscopy, cells were seeded on glass bottom #1.5H µ-slides (IBIDI, Germany) or glass bottom #1.5H-N 96-well plates (Cellvis, USA). Imaging was performed on a Nikon Widefield Ti2 equipped with a sCMOS, PCO.edge camera and a live-cell incubator. Image acquisition was done in Flurobrite medium (GIBCO) supplemented with 2% FBS, 1:100 PenStrep, and 1x GlutaMax at 37 °C and 5% CO₂. The following light sources (LEDs) and emission filters were used for the different channels: CFP (cerulean): excitation 438/29, emission 473/24 nm; GFP (dClover2): excitation 475/28 nm, emission 520/26 nm; YFP (mClover3, dClover2): excitation 511/16 nm, emission 540/30 nm; RFP (mScarlet-I) excitation 555/28 nm, emission 642/80 nm. Objectives: 40x ApoFluor, NA 0.95, WD 250 µm; 20x Plan Apo, NA 0.8, WD 1 mm. Illumination time for CFP was usually 500 ms and 2 s for all other channels. To synchronize the circadian rhythms, cells were either washed twice with cold PBS for 2 min (Fig. 1f, Supplementary Figs. 3 and 7a) or treated with 1 µM dexamethasone for 30 min followed by washing with warm PBS (Figs. 2–4), or by exchange to pre-warmed imaging medium (Supplementary Figs. 5 and 7b, c). Imaging started 2 h after synchronization with a regular imaging interval of 1 h.