The ability to simultaneously assess every gene in a genome for a role in a particular process has obvious appeal. This protocol describes how to perform genome-scale RNAi library screens in bloodstream-form African trypanosomes, a family of parasites that causes lethal human and animal diseases and also serves as a model for studies on basic aspects of eukaryotic biology and evolution. We discuss strain assembly, screen design and implementation, the RNAi target sequencing approach and hit validation, and we provide a step-by-step protocol. A screen can yield from one to thousands of 'hits' associated with the phenotype of interest. The screening protocol itself takes 2 weeks or less to be completed, and high-throughput sequencing may also be completed within weeks. Pre- and post-screen strain assembly, validation and follow-up can take several months, depending on the type of screen and the number of hits analyzed.
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We thank P. Englund, Z. Wang, J. Morris and M. Drew ('the pZJM library team') for the RNAi plasmid library, B. Dujon for the pSCM525 plasmid containing a human-codon–optimized version of the I-SceI gene and S. Obado for contributions to the assembly of the 2T1:T7 T. brucei strain. We also thank B. Brunk and O. Harb, and the GeneDB team for making RIT-seq data available through the GeneDB and TritrypDB databases. The work was funded by grants from The Wellcome Trust; 093010/Z/10/Z (D.H.), 100476 (Strategic Award to Biological Chemistry and Drug Discovery, Dundee), 100320/Z/12/Z (D.H. Senior Investigator Award) and 085775/Z/08/Z (The Wellcome Trust Sanger Institute).
The authors declare no competing financial interests.
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Glover, L., Alsford, S., Baker, N. et al. Genome-scale RNAi screens for high-throughput phenotyping in bloodstream-form African trypanosomes. Nat Protoc 10, 106–133 (2015). https://doi.org/10.1038/nprot.2015.005
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