RNA FISH was performed as described previously (Chen et al., 2017 (link)). Cells were fixed with 4% formaldehyde in PBS for 15 min at room temperature, followed by permeabilization with 0.5% Triton X‐100, 2 mM VRC (NEB) on ice, and two times 2x SSC wash for 10 min each. Probes were first amplified with PCR using PAM expression plasmid in RNA pulldown experiment. PCR products were then precipitated by ethanol, nick‐translated and labelled with Green d‐UTP (Abbott) and nick translation kit (Abbott). For each FISH experiment, 200 μg of probe and 20 μg of yeast tRNA were lyophilized and redissolved in 10 μl formamide (Ambion), followed by denaturation at 100°C for 10 min and chilled immediately on ice. Denatured probes were mixed with hybridization buffer at 1:1 ratio. 20 μl of hybridization mix was added onto fixed cells, followed by putting coverslip on it and incubated at 37°C for 16 h in a humidified chamber. Cells were then washed twice in 2x SSC, 50% formamide; thrice in 2x SSC; and once in 1x SSC for 5 min each in 42°C. Cells were mounted by coverslip with ProLong Gold Antifade Reagent with DAPI (Invitrogen). Fluorescence images were taken in Olympus microscope FV10000 and FV10‐ASW software (version 01.07.02.02, Olympus).
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