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

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Africa
  • Gene Library
  • Genetic Testing / methods*
  • Genome, Protozoan / genetics*
  • Phenotype*
  • RNA Interference*
  • Trypanosoma / genetics*