domingo, 28 de diciembre de 2014

Genome-scale RNAi screens for high-throughput phenotyping in bloodstream-form African trypanosomes : Nature Protocols : Nature Publishing Group

Genome-scale RNAi screens for high-throughput phenotyping in bloodstream-form African trypanosomes : Nature Protocols : Nature Publishing Group







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

Nature Protocols
 
10,
 
106–133
 
 
doi:10.1038/nprot.2015.005
Published online
 

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.


  1. Overview of RIT-seq.
    Figure 1
  2. Example outputs from RIT-seq.
    Figure 2
  3. Overview of the protocol.
    Figure 3
  4. Strains and constructs for RNAi library screening and hit validation.

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