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

    32 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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