smFISH image acquisition and analysis

smFISH images were taken on a Nikon TiE microscope with a CFI HP TIRF objective (100x, NA 1.49, Nikon), and an EMCCD (Andor, iXon Ultra 888). Nikon TiE epifluorescent microscope. Samples were excited using the 647nm laser (Cobolt MLD) (~15–20 mW for 200–300ms), poly-A FISH was imaged using the 561nm laser (Coherent Obis) (~15–20 mW for 200–300ms), and Pab1-Halotag signal was imaged with a 488nm laser (Cobolt MLD) (~10–15 mW for 200–300 ms), and DAPI (CL2000, Crystal Laser) (~5–10 mW for 100 ms). Imaging of the nucleus was done using the 405nm laser and DIC images were taken as well. Z-stacks of 21 planes, 2uM thick were obtained. Images were analyzed using FISH-quant¹⁰⁶. Briefly, RNA spots were identified using big fish. For the smFISH colocalization analysis, RNA spot intensities were normalized by dividing by the mean intensity of each cell. For each RNA spot, the mean Pab1 intensity in a 3×3 pixel square around the centroid was calculated. The Pab1 intensity was then measured for 100 random locations in the cell in 3×3 pixel locations. Finally, a distribution was calculated for both the random Pab1 signal and the Pab1 signal that corresponds to a RNA spot. The Z-score of the mean intensity of the Pab1 signal in a RNA spot compared to the Pab1 signal in a random spot was compared, and this is termed the ‘colocalization score’. Each Z-score is calculated independently for each cell, and the average shown is for every cell.