To control for effects due to non-specific effects of shRNAs, we profiled 27 distinct negative control hairpins which do not have a known target in the cell. These hairpins provide a measurement of the variability of the expression profiles due to random variability or triggered by ‘off-target’ effects of the shRNA lentiviruses. Assuming that any observed effects in the negative control hairpins are due to ‘off-target’ effects and observed effects in the targeting hairpins include a mix of both ‘off-target’ effects and ‘on-target’ effects, we use permutations of the negative controls to assign a false discovery rate (FDR) confidence level for being an ‘on-target’ hit to each gene. As such, a gene would only reach genome-wide significance if the number of genes and scale of the effect was much larger than would be observed randomly among all of the expression changes found for the negative control hairpin.
