Measurement of knockdown efficiency for hit verification. (a) RT-qPCR results of all the hits identified in both replicates after knockdown. An enlarged graph of the bottom part of the original graph is also included. Cells (hTERT-RPE1 dCas9-KRAB-BFP PA-mCherry H2B-mGFP) were infected with corresponding sgRNAs (Table S4) and puromycin selected for 3 d before harvesting. Harvested cells were split, and one was used for RNA extraction and RT-qPCR analysis to measure the percentage of knockdown (KD percentage), while the other half was used for FACS analysis to measure the percentage of sgRNA infection (Infection percentage; Fig. S5 b). ACTB was used as an internal control to normalize the variability on expression levels. Error bar: SD between triplicates. AU, arbitrary units. (b) Knockdown efficiency (KD efficiency) of all the hits identified in both replicates. Knockdown percentage (KD percentage) was measured based on RT-qPCR results. BFP was coexpressed on the sgRNA construct, and only cells with BFP intensity above a threshold value determined by control cells were considered successfully infected cells. Percentage of successful infection (Infection percentage) was measured by FACS and for each gene, and KD efficiency was calculated using KD percentage divided by its corresponding infection percentage.