Fig. 2

Gating strategy for defining infected cells and screen performance. (A) SARS-CoV-2 N protein average intensity was used to classify each cell as infected or uninfected. The maximum intensity cut-off for uninfected cells was calculated as the median + (2 x IQR) of the uninfected cell population. Cells with a SARS-CoV-2 mean intensity above this value were classified as infected. The per-cell SARS-CoV-2 mean intensities for negative control conditions (uninfected, mock, siOTP-NT) are shown in the plot. The calculated cut-off is indicated by the vertical dotted line (>0.005). (B) Mimic and inhibitor screen replicate plate reproducibility was assessed for cell count and virus infection using correlation analysis. Cell count (left panels) and % cells infected (right panels) are both represented as fold change (FC) to siOTP-NT and replicate plates for all miRNA samples are plotted. The Pearson Correlation Co-efficient is displayed for each comparison. (C) Box and whisker plots showing distribution of positive (siPLK1, siTOX) and negative (mock, siOTP-NT, uninfected) controls for cell count (left panels) and % cells infected (right panels) for all wells on all plates in both screens. Outlier wells are defined as having values > 1.5 x IQR above the 75^th percentile or < 1.5 x IQR below the 25^th percentile and are represented by black dots in the plot. Note, knock down of genes that induce extensive cell death, such as the viability positive control PLK1, are associated with higher variability in terms of infection due to the limited number of cells remaining in the well.