We infected each shRNA into ESCs, isolated RNA after 4 days, and profiled their effects on global transcription by hybridization to genome-wide microarrays (Figure 1a, see Methods). We employed a stringent procedure to control for non-specific effects due to viral infection, generic RNAi responses, or ‘off-target’ effects. Expression changes were deemed significant only if they exceeded the maximum levels observed in any of the negative controls, showed a two-fold change in expression compared to the negative controls, and had a low false discovery rate (FDR) assessed across all genes based on permutation tests (Figure 1b, see Methods). This approach controls for the overall rate of non-specific effects by estimating the number and magnitude of observed effects in the negative control hairpins, where all effects are non-specific.
