Abstract
This protocol describes the various steps and considerations involved in planning and carrying out RNA interference (RNAi) genome-wide screens in cultured Drosophila cells. We focus largely on the procedures that have been modified as a result of our experience over the past 3 years and of our better understanding of the underlying technology. Specifically, our protocol offers a set of suggestions and considerations for screen optimization and a step-by-step description of the procedures successfully used at the Drosophila RNAi Screening Center for screen implementation, data collection and analysis to identify potential hits. In addition, this protocol briefly covers postscreen analysis approaches that are often needed to finalize the hit list. Depending on the scope of the screen and subsequent analysis and validation involved, the full protocol can take anywhere from 3 months to 2 years to complete.
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Acknowledgements
We thank all members of the Perrimon laboratory as well as all past and present DRSC screeners for sharing their experience with us. None of the protocols described here would have been possible without their contributions. Work at the DRSC is supported by grant R01 GM067761 from the NIGMS. N.P. is an investigator of the Howard Hughes Medical School.
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Ramadan, N., Flockhart, I., Booker, M. et al. Design and implementation of high-throughput RNAi screens in cultured Drosophila cells. Nat Protoc 2, 2245–2264 (2007). https://doi.org/10.1038/nprot.2007.250
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DOI: https://doi.org/10.1038/nprot.2007.250
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