MCF7 viability screen performance in different pool sizes. (a) Scatter plots of 2,000 hairpins common to the 2,000, 5,000 and 10,000 shRNA pools showing high correlation of normalized scores (median of four replicates) between different pool sizes. Numbers indicate Pearson correlation between pools. (b) Plot of observed barcode screen log ratios for validated positive and negative controls in the 2,000, 5,000 and 10,000 shRNA pools versus expected scores based on single hairpin GFP competition assay scores. Positive controls are in blue and negative controls are in red. The horizontal dotted line indicates the threshold used for hit calling in the screen. Based on this threshold, 5 out of 6 valid positive controls were called hits whereas 0 out of 11 negative controls did not score as hits. (c) Distribution of log ratios of 101 non-targeting hairpins in the 2k pool. (d) Scatter plots of z-scores from four biological replicates of the 10k pool MCF7 viability screen, indicating a good agreement between replicates. Numbers indicate Pearson correlation between replicates.