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

Nature Protocols volume 10, pages 106–133 (2015)Cite this article

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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

Authors and Affiliations

  1. Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, UK

    Lucy Glover, Nicola Baker, Sebastian Hutchinson & David Horn

  2. London School of Hygiene and Tropical Medicine, London, UK

    Sam Alsford

  3. Oxford Nanopore Technologies, Oxford, UK

    Daniel J Turner

  4. Institute of Biotechnology, Universidad Nacional Autónoma de México, Cuernavaca, México

    Alejandro Sanchez-Flores

  5. Centre for Genomic Research, Institute of Integrative Biology, University of Liverpool, Liverpool, UK

    Christiane Hertz-Fowler

  6. The Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.,

    Matthew Berriman

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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.

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Correspondence to David Horn.

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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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