Figure 3.

RNAi screening workflow. dsRNA libraries synthesized by in vitro transcription (IVT) of PCR amplicons using T7 RNA polymerase. RNAs are then plated into microtiter plates, usually using liquid handling robots. Bathing or reverse transfection is performed by directly plating cells on top of spotted dsRNA. Depending on the specific experimental setup, an additional dsRNA (co-RNAi), treatment, or condition (chemogenetics) can be applied to the cells in consecutive steps. Each plate is assayed using, for example, biochemical readouts, signaling reporter assays, or microscopy to measure the resulting phenotype.