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Nat Protoc. 2015 Jan;10(1):106-33. doi: 10.1038/nprot.2015.005. Epub 2014 Dec 11.

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

Author information

1
Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, UK.
2
London School of Hygiene and Tropical Medicine, London, UK.
3
Oxford Nanopore Technologies, Oxford, UK.
4
Institute of Biotechnology, Universidad Nacional Autónoma de México, Cuernavaca, México.
5
Centre for Genomic Research, Institute of Integrative Biology, University of Liverpool, Liverpool, UK.
6
The Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.

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.

PMID:
25502887
DOI:
10.1038/nprot.2015.005
[Indexed for MEDLINE]

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