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Genome-scale RNAi screens for high-throughput phenotyping in bloodstream-form African trypanosomes

Abstract

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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Figure 1: Overview of RIT-seq1.
Figure 2: Example outputs from RIT-seq1,2.
Figure 3: Overview of the protocol.
Figure 4: Strains and constructs for RNAi library screening and hit validation.
Figure 5: Workflow of high-throughput RIT-seq (Steps 39–55).

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Acknowledgements

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).

Author information

Affiliations

Authors

Contributions

L.G., S.A. and D.H. set up components for, and assembled, the T. brucei RNAi libraries; S.A., L.G., N.B. and D.H. carried out RNAi screens; D.J.T., C.H.-F., M.B. and D.H. conceived the Illumina RIT-seq approach; D.J.T. carried out Illumina DNA sequencing; A.S.F., C.H.-F. and M.B. developed protocols for sequence mapping and analysis; A.S. carried out sequence mapping and analysis; S.H. developed our more recent protocols for sequence mapping and analysis; S.A., L.G. and N.B. characterized hits; L.G., S.A., D.J.T., N.B., A.S.F. and S.H. wrote the protocols; and D.H. wrote other sections of the paper.

Corresponding author

Correspondence to David Horn.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Method 1

Additional details regarding routine handling of Trypanosoma brucei. (PDF 56 kb)

Supplementary Method 2

RITseq.py — Python script for sequence mapping. (ZIP 1 kb)

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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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