Extended Data Fig. 3: Extended data comparing dMCP and HCR single-molecule RNA puncta.
(a-c) Maximum intensity projection confocal microscopy images of fixed U2OS cells expressing H2B-mCherry-24xMS2 RNA tagged by 4xmNG-HA-dMCP and HCR probes targeting mCherry. dMCP signal is shown on the leftmost column (green), HCR signal shown left-of-center (magenta), merged images are shown right-of-center, and an overlay with digitally-labeled cytosolic spots as determined by ImageJ’s particle analysis plug-in is shown on the rightmost column. In the overlayed images, green spots are dMCP spots that do not overlap HCR, magenta spots are HCR spots that do not overlap dMCP, and white spots are spots from both channels which overlap. H2B-mCherry signal is shown in the bottom right. (d) Signal-to-noise ratio (SNR) of dMCP spots compared with HCR spots across three cells. SNR is measured as the average intensity of a spot divided by the average intensity of the entire cytosolic region excluding spots. Both values are calculated with the average background intensity of the local culture medium, calculated as the MFI of a large boxed region in each image in which no cells were detected, subtracted. Bars represent the mean of each population. (c) Area in square micrometers of dMCP spots in comparison to HCR spots. Bars represent the mean of each population. (d) Tabulated values for co-localization between dMCP and HCR spots. dMCP spot counts per cell are a; n = 182, b: n = 299, c: n = 447. HCR spot counts per cell are a: n = 138, b: n = 269, c: n = 345. The lower HCR spot count is primarily due to closely spaced, large puncta that were collectively counted as single foci because they could not be individually resolved.
