Quantifying Cellular RNA using smRNA FISH

The smRNA FISH protocol is based on previously published ones^{4 (link)}. Briefly, cells were grown on uncoated 18 mm × 18 mm #1.5 coverslips, fixed with 4% formaldehyde in 1X PBS for 10 minutes, washed in 1X PBS and stored overnight in 70% ethanol at 4 °C. After rehydration, hybridization with probes at final concentrations of 62 nM was carried out in 2X SSC/10% formamide/10% dextran sulfate for 6–14 hours at 37 °C. The hybridization mix also contained 10 mM ribonucleoside vanadyl complex (New England Biolabs) and in some experiments 100–200 μg/ml of yeast tRNA to protect cellular RNAs. After washing with 10% formamide/2X SSC for 30 minutes at 37 °C and twice with 2X SSC for 5 minutes at room temperature, samples were stained with 4′,6-diamidino-2-phenylindole (DAPI, 1 μg/ml in 2X SSC) and mounted in Prolong Gold (ThermoFisher/Life Technologies). Slides were cured at room temperature for 2–3 days before imaging. Oligonucleotide (20-mer) probes against human POLR2A (n = 48, Supplementary Table S1), the first intron of human POLR2A (n = 47, Supplementary Table S2) and human TFRC labeled at the 3′ end with Quasar 670 (λ_ex = 646 nm, λ_em = 670 nm), Quasar 570 (λ_ex = 548, λ_em = 566) and Quasar 570 dyes, respectively, were purchased from BioSearch Technologies. All experiments were performed at least in duplicates.