Single‐molecule RNA fluorescence in situ hybridisation (smRNA FISH)

Cells were plated on 6‐well plates containing 18 mm round cover glasses (Fisher Scientific) treated with a solution of 25 μg/ml Fibronectin (Sigma‐Aldrich) diluted in 1× PBS for 30 min at room temperature. Wells were seeded with 0.2 × 10⁶ cells. Cells were cultured in serum‐free DMEM (Gibco), supplemented with 1% PSG antibiotics, to prevent cell division. Ten hours later, cells were treated with 100 nM dexamethasone (Sigma‐Aldrich) for 30 min. The treatment was followed by medium change. For the experiment where the circadian clock was synchronised using temperature cycles, the same procedure was applied except entrained cells were not treated with dexamethasone. The smRNA FISH protocol was largely adapted from the Stellaris RNA FISH protocol for adherent cells, which describes the method used by Raj et al (2008). Stellaris exonic probes coupled with Quasar570 (548/566nm, Red, BMAL1^Red, NR1D1^Red) or Quasar670 (647/670nm, FarRed, BMAL1^FarRed, CRY1^FarRed) were hybridised to targeted mRNA. Probe sets are shown in Table EV1. The hybridisation was performed in 50 μl of Stellaris hybridisation buffer complemented with 250 μg/ml Yeast tRNA (Ambion) and 5 mM Ribonucleoside Vanadyl Complex (New England Biolabs). Cells and nuclei were then co‐strained with 0.4 μg/ml green HCS CellMask (Invitrogen) and 0.7 μg/ml DAPI (Thermo Fisher), respectively. HCS CellMask and DAPI were diluted in the Stellaris wash buffer. Cover glasses were finally mounted onto microscopy slides in ProLong™ Gold Antifade Mountant (Thermo Fisher). For each time point in both time course experiments (4 and 6 h sampled experiments), cells were plated in triplicate. Each replicate was independently synchronised and prepared for smFISH analysis.