Figure 2

Performance of shRNA screen. (A) Heatmap representation of unsupervised hierarchical cluster analysis of screen data. Columns represent screened cell lines (key provided), and rows represent the 558 different shRNAs (here, mean-centered). The color scale indicates fold-depletion (blue) or fold-enrichment (red) of shRNAs in T = 4 week vs. T = 0 comparisons. Note that replicate screens for each cell line tend to cluster together, which demonstrates reproducibility across all steps of the screen (infection, cell line passaging, PCR amplification, and microarray hybridization). The basis for the single outlier (Capan1, far right) is unclear. (B) Time course analysis of shRNA depletion/enrichment, shown for a single cell line (HPAC). Note that across the time series, library shRNAs show expected increased depletion (blue, top of heat map) or enrichment (red; bottom of heat map), consistent with competitive growth selection. (C) Depletion/enrichment status of selected genes is shown. Knockdown of the same gene by different shRNAs often produced concordant phenotypes; e.g., Pearson correlation (R) = 0.903 for two hairpins targeting NOL3, and R = 0.995 for two hairpins targeting PLSCR1. (D) Frequency plot of observed Pearson correlations (R values; correlating depletion/enrichment vectors across the cell line panel) between hairpin pairs targeting the same gene (dark gray bars) compared to a null distribution of Pearson correlations generated from randomly permuted data (light gray bars). Note, the rightward shift of observed Pearson correlations, above that expected by chance, reflects enrichment of on-target shRNAs/phenotypes.