Genome-Wide RNAi Screens for RNA Processing Events in Drosophila melanogaster S2 Cells

Over the past 10 years, the design and application of genome-wide screening (GWS) has improved to the point that it can now be done at level of the individual laboratory. The advantages of GWSs compared to classical genetic screens include: immediate identification of a positive scoring gene, relatively short period of time necessary to conduct the screen (as little as 1 week), cell lines do not present developmental needs for gene expression that an organism normally would, and validation/confirmation of results is straightforward. Here, we describe a general protocol for GWS to be conducted in Drosophila melanogaster S2 cells. We provide specific details on what type of experiments must be done before initiating a screen, the materials that are required to conduct a screen, and make suggestions on methods to carry out secondary screening and counter-screening once the initial GWS is complete. Multiple considerations are also raised that focus on how to anticipate false positives/negatives and how to minimize their occurrence through intelligent design. Finally, we provide specific examples of data that our group has gathered from published genome-wide screens in order to exemplify how "hits" are scored and confirmed.