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Abstract

The development of advanced functional genomic tools has paved the way for systematic investigations of biological processes in health and disease. In particular, the implementation of RNA interference (RNAi) as a genome-wide, loss-of-function screening tool has enabled scientists to probe the role for every gene in cellular assays and many new factors for various processes have been discovered employing RNAi screens in recent years. However, the results also demonstrate the complexity of biological systems and indicate that we are still a long way from understanding functional networks in depth. Nevertheless, RNAi screens present a powerful method to interrogate gene function in high-throughput and different methods to elicit RNAi in mammalian cells have been developed. Here, we describe steps that should be considered when planning an RNAi screen employing endoribonuclease prepared (e)siRNAs. We provide useful information on how to implement the screen and analyze the results. Furthermore, we discuss strategies for hit validation and present an outline on how to follow-up on verified hits to gain a molecular understanding of the underlying phenotypes.