title: 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.
authors.affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, UK.
authors.forename: Lucy
authors.initials: L
authors.lastname: Glover
authors.affiliation: London School of Hygiene and Tropical Medicine, London, UK.
authors.forename: Sam
authors.identifier: 0000000186214920
authors.initials: S
authors.lastname: Alsford
authors.affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, UK.
authors.forename: Nicola
authors.initials: N
authors.lastname: Baker
authors.affiliation: Oxford Nanopore Technologies, Oxford, UK.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Turner
authors.affiliation: Institute of Biotechnology, Universidad Nacional Autónoma de México, Cuernavaca, México.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Sanchez-Flores
authors.affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, UK.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Hutchinson
authors.affiliation: Centre for Genomic Research, Institute of Integrative Biology, University of Liverpool, Liverpool, UK.
authors.forename: Christiane
authors.initials: C
authors.lastname: Hertz-Fowler
authors.affiliation: The Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.
authors.forename: Matthew
authors.identifier: 0000000295810377
authors.initials: M
authors.lastname: Berriman
authors.affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, UK.
authors.forename: David
authors.initials: D
authors.lastname: Horn
mesh_terms: D000349:Africa; D015723:Gene Library; D005820:Genetic Testing / Q000379:methods*; D018503:Genome, Protozoan / Q000235:genetics*; D010641:Phenotype; D034622:RNA Interference; D014345:Trypanosoma / Q000235:genetics*