Analysis of MED1 IF with RNA FISH

3D image data gathered in RNA FISH and IF channels for ~120 cells per FISH probe (gene) was processed with custom Python and MATLAB scripts as described previously^59,60. Briefly, FISH foci were manually identified in individual z stacks through intensity thresholds, centered along a box of size l = 1 μm, and stitched together in 3-D across z stacks. Only cells with 1 or 2 FISH foci were considered for downstream analyses. For every RNA FISH focus identified, signal from the corresponding location in the IF channel is gathered in the l x l square centered at the RNA FISH focus at every corresponding z-slice. The IF signal centered at FISH foci for each FISH and IF pair are then combined and an average intensity projection is calculated, providing averaged data for IF signal intensity within a l x l square centered at FISH foci. The same process was carried out for the FISH signal intensity centered on its own coordinates, providing averaged data for FISH signal intensity within a l x l square centered at FISH foci. As a control, this same process was carried out for IF signal centered at randomly selected nuclear positions within the nucleare volume determined from DAPI staining through the z stack image as described in detail in ref. ⁵⁹. Average MED1 IF intensity projections centered at FISH foci were visualized using the same intensity-color range for all genes, ranging from minimal to maximal observed IF intensity within the 1 μm x 1 μm area (Fig. 3h, Extended Data Fig. 5f). For quantitative comparison of MED1 IF signal between different genes (Fig. 3i), MED1 IF signal at each FISH focus was normalized to the average signal at random spots from the same dataset to account for the difference in overall MED1 IF intensity between different datasets.