Fig. 6: Performance of single-transcript methods can be attributed to RNA labeling strategy.

a Performance of single-transcript methods was compared using cells that shared the same steps of the measurement process except for the RNA labeling step. b Resolvability of FISH and HCR was assessed by plotting AUC calculated from adjacent stimulus levels. Pairs of adjacent IPTG concentrations are shown as numbers within the plots, as indicated in figure legend. c A two-state promoter model was used to evaluate transcription kinetics. d Negative binomials were used to fit single-transcript distributions for FISH (blue) and HCR (dark orange). e Estimates of burst frequency are plotted for FISH versus HCR. The RNA lifetime was assumed to be a constant (2.8 min). f Estimates of burst size, and g estimates of burst size after correcting for hybridization efficiency, are plotted for FISH versus HCR. For parts (f) and (g), burst size is the number of transcripts per burst. For parts (e), (f), and (g), the scatter plot numbers 2, 3, 4, 5, 6, 7, and 8 represent 5, 10, 20, 40, 100, 400, and 1000 μmol/L IPTG, respectively. The 0 μmol/L IPTG case is not shown, in order to more easily see the trend for the remaining induction conditions. For (b), (e), (f), and (g): Diagonal line indicates equivalent performance between the two methods. Color indicates biological replicates one (orange), two (green), and three (purple), as indicated in the figure legend. Large gray numbers in the top-left and bottom right-corners indicate how many values were higher for the method plotted on the y-axis or x-axis, respectively. The two-sided p-values for a sign test are shown within each plot.