Design and implementation of high-throughput RNAi screens in cultured Drosophila cells.
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.
Ramadan
Nadire
N
Department of Genetics and Drosophila RNAi Screening Center, Harvard Medical School, Boston, Massachusetts 02115, USA.
Flockhart
Ian
I
Booker
Matthew
M
Perrimon
Norbert
N
Mathey-Prevot
Bernard
B
D000818:Animals; D016477:Artifacts; D002478:Cells, Cultured; D004330:Drosophila / Q000166:cytology / Q000235:genetics*; D015723:Gene Library; D023281:Genomics / Q000379:methods*; D016133:Polymerase Chain Reaction; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000737:chemistry
D012330:RNA, Double-Stranded