Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, UK.
London School of Hygiene and Tropical Medicine, London, UK.
Nat Protoc. 2015 Jan;10(1):106-33. doi: 10.1038/nprot.2015.005. Epub 2014 Dec 11.
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.
同时评估基因组中每个基因在特定过程中的作用的能力具有明显的吸引力。本协议描述了如何在血液形式的非洲锥虫中进行全基因组 RNAi 文库筛选，非洲锥虫是一类寄生虫，会导致致命的人类和动物疾病，也可作为研究真核生物生物学和进化基本方面的模型。我们讨论了菌株组装、筛选设计和实施、RNAi 靶序列测序方法和命中验证，并提供了一个逐步的方案。筛选可以产生一个到数千个与感兴趣表型相关的“命中”。筛选方案本身需要 2 周或更短的时间完成，高通量测序也可以在几周内完成。筛选前和筛选后的菌株组装、验证和后续工作可能需要几个月的时间，具体取决于筛选的类型和分析的命中数量。
