Next-generation sequencing for high-throughput RNA interference screens

Abstract

Ribonucleic acid interference (RNAi) screening has emerged as an indispensable genetic research tool, allowing determination of phenotypic effects after silencing entire suites of genes. As the catalog of fully sequenced genomes and transcriptomes grows, production of small interfering/short-hairpin RNA libraries that target every gene in a particular cell, tissue, or organism is achievable, allowing high-throughput “genome-wide” RNAi screening. This technology has been embraced by cancer biologists and has been used to analyze a myriad of phenotypic effects of genetic loss of function in human cancers. A basic RNAi screening scheme includes silencing of a panel of genes in a cell population, followed by identification of a phenotypic change (in cancer research, this might include cell death, cell invasion, sensitivity to therapeutics). Upon identification of cells exhibiting the phenotype of interest, it is necessary to determine which specific shRNAs are responsible. Previously, this process was laborious, requiring tedious DNA extraction, PCR amplification, and individual cloning and sequencing of PCR amplicons to determine the specific shRNA(s) harbored by cells. With the advent of next-generation sequencing (NGS), identification of individual shRNAs harbored by cells has been revolutionized. NGS allows rapid and specific identification of shRNA oligomers present in the cell(s) of interest and requires minimal amounts of source material. This chapter will describe the use of NGS in RNAi screens with a focus on cancer biology and provide resources for those interested in pursuing NGS-powered RNAi screens.