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

Lucy Glover, Sam Alsford, Nicola Baker, Daniel J. Turner, Alejandro Sanchez-Flores, Sebastian Hutchinson, Christiane Hertz-Fowler, Matthew Berriman, David Horn

    Research output: Contribution to journalArticle

    18 Citations (Scopus)

    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.

    Original languageEnglish
    Pages (from-to)106-33
    Number of pages28
    JournalNature Protocols
    Volume10
    Issue number1
    DOIs
    Publication statusPublished - Jan 2015

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    Trypanosomiasis
    RNA Interference
    Genes
    Throughput
    Genome
    Animal Diseases
    Libraries
    Parasites
    Phenotype
    Screening
    Animals

    Cite this

    Glover, L., Alsford, S., Baker, N., Turner, D. J., Sanchez-Flores, A., Hutchinson, S., ... Horn, D. (2015). Genome-scale RNAi screens for high-throughput phenotyping in bloodstream-form African trypanosomes. Nature Protocols, 10(1), 106-33. https://doi.org/10.1038/nprot.2015.005
    Glover, Lucy ; Alsford, Sam ; Baker, Nicola ; Turner, Daniel J. ; Sanchez-Flores, Alejandro ; Hutchinson, Sebastian ; Hertz-Fowler, Christiane ; Berriman, Matthew ; Horn, David. / Genome-scale RNAi screens for high-throughput phenotyping in bloodstream-form African trypanosomes. In: Nature Protocols. 2015 ; Vol. 10, No. 1. pp. 106-33.
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    Glover, L, Alsford, S, Baker, N, Turner, DJ, Sanchez-Flores, A, Hutchinson, S, Hertz-Fowler, C, Berriman, M & Horn, D 2015, 'Genome-scale RNAi screens for high-throughput phenotyping in bloodstream-form African trypanosomes', Nature Protocols, vol. 10, no. 1, pp. 106-33. https://doi.org/10.1038/nprot.2015.005

    Genome-scale RNAi screens for high-throughput phenotyping in bloodstream-form African trypanosomes. / Glover, Lucy; Alsford, Sam; Baker, Nicola; Turner, Daniel J.; Sanchez-Flores, Alejandro; Hutchinson, Sebastian; Hertz-Fowler, Christiane; Berriman, Matthew; Horn, David.

    In: Nature Protocols, Vol. 10, No. 1, 01.2015, p. 106-33.

    Research output: Contribution to journalArticle

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    Glover L, Alsford S, Baker N, Turner DJ, Sanchez-Flores A, Hutchinson S et al. Genome-scale RNAi screens for high-throughput phenotyping in bloodstream-form African trypanosomes. Nature Protocols. 2015 Jan;10(1):106-33. https://doi.org/10.1038/nprot.2015.005