To assess the effects of a lincRNA on gene expression, we profiled the changes in gene expression after knocking down each lincRNA gene. Specifically, for each lincRNA with at least 1 validated hairpin we profiled the genome-wide expression level changes after knockdown across 2 independent infections (see above). To control for expression changes due to viral infection, we performed five independent infections containing no RNAi hairpin (pLKO.5-nullT). This control hairpin was embedded in each RNA prep plate. To control for effects due to an off-target RNAi effect, we profiled 27 distinct negative control hairpins which do not have a known target in the cell. These hairpins included 6 RFP hairpins, 10 GFP hairpins, 6 Luciferase hairpins, and 5 LacZ hairpins. These hairpins provide a measurement of the variability of the RNAi response triggered due to non-specific effects. Furthermore, we profiled hairpins targeting 147 lincRNAs, including 10 with a second best hairpin, and 40 protein-coding genes in biological replicate. The hairpins and their replicates were randomly distributed across 7 96-well plates and prepared in batches. Each RNA preparation batch contained 1
