Fig 2. SCRINSHOT specificity relies on stringent hybridization of padlock probes to their target RNAs.

Images of SCRINSHOT signal, using normal Scgb1a1 padlock probe (A), a Scgb1a1 padlock probe with a point mutation at its ligation site (B), a Scgb1a1 padlock probe with 3′-scrambled arm (C) and normal padlock probe but omitting SplintR ligase (D). Actb normal padlock probe was used in all conditions as internal control. DAPI: blue, Scgb1a1: gray, Actb: red. “n” indicates the number of airway cells in the corresponding images. (A’-D’) Magnified areas of the indicated positions (square brackets) of images in the left. Pink outlines show the 2-μm expanded airway nuclear ROIs, which are considered as cells. Scale bar: 150 μm. (E) Violin plot of the Scgb1a1 and Actb signal dots ratio in all airway cells. The ratio of cells with zero Actb-dots was considered as zero. There were no statistically significant differences for Actb signal dots, between the analyzed conditions, except for the SplinR^neg, that gave no signal, as expected, indicating that ligation is obligatory for signal detection (P values for Actb dot comparisons: "SplintR^pos versus mismatch" = 0.7706, “SplintR^pos versus scrambled" = 0.8837, "SplintR^pos versus SplintR^neg" ≤ 0.0001, "mismatch versus scrambled" = 0.6523, "mismatch versus SplintR^neg" ≤ 0.0001, "scrambled versus SplintR^neg" ≤ 0.0001). (F) Violin plot of the Scgb1a1 and Actb fluorescence intensity ratio in all airway cells. SplintR^pos n = 473, mismatch n = 574, 3´-scrambled n = 507 and SplintR^neg n = 488. The data underlying this Figure can be found in 10.5281/zenodo.3634561. ROI, region of interest.