Analysis of γ-Tubulin and α-Tubulin signals in the secondary screen. (A) Representative images of unmodified U-2 OS cells with different numbers of detected γ-Tubulin puncta (from population of NTC controls) and distribution of γ-Tubulin puncta numbers in cells treated with nocodazole (Noc) or the indicated crRNA pools. Line charts show normalized data (rZ normalization, central reference = NTC) and pie charts show raw data (percentage of cells). Scale bar, 50 µm. (B) Scatterplot of normalized values (rZ normalization, central reference = NTC) for the proportion of cells with 1 versus 0 detected γ-Tubulin punctum. crRNAs with values to the left of the vertical −2.5*SD (NTC) cut-off line were classed as causing a decrease in the proportion (prop.) of cells with one detected γ-Tubulin punctum (243/377 crRNA pools). This phenotype could be associated with either an increase in the proportion of cells with >1 MTOC (193 crRNAs; values below the horizontal 2.5*SD [NTC] cut-off line; regions shaded in pink and dark pink) or an increase in the proportion of cells with no MTOC (50 crRNAs; values above the horizontal 2.5*SD [NTC] cut-off line; region shaded in blue). An additional threshold based on crLIS1 controls (−2.5* SD) was introduced for the one punctum feature to categorize crRNAs that yielded a stronger phenotype than crLIS1. (C) Quantification of α-Tubulin morphology in U-2 OS PEX cells generated by linear discriminant analysis (rZ, central reference = NTC) and representative immunofluorescence images of examples (labeled in gold text in the plot). In B and C, library copies of crPLK1 and crLIS1 are labeled with “(L).” Scale bar, 25 µm. Data points in A–C represent the mean values of two independent experiments with at least four wells per crRNA per experiment. Hits were defined as exceeding ± 2.5*SD of (A–C) NTC and/or (B) crLIS1. See Table S6 for full dataset.