Open Access Highly Accessed Method

High-throughput RNA interference screening using pooled shRNA libraries and next generation sequencing

David Sims, Ana M Mendes-Pereira, Jessica Frankum, Darren Burgess, Maria-Antonietta Cerone, Cristina Lombardelli, Costas Mitsopoulos, Jarle Hakas, Nirupa Murugaesu, Clare M Isacke, Kerry Fenwick, Ioannis Assiotis, Iwanka Kozarewa, Marketa Zvelebil, Alan Ashworth* and Christopher J Lord*

Author Affiliations

The Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK

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Genome Biology 2011, 12:R104  doi:10.1186/gb-2011-12-10-r104

Published: 21 October 2011

Abstract

RNA interference (RNAi) screening is a state-of-the-art technology that enables the dissection of biological processes and disease-related phenotypes. The commercial availability of genome-wide, short hairpin RNA (shRNA) libraries has fueled interest in this area but the generation and analysis of these complex data remain a challenge. Here, we describe complete experimental protocols and novel open source computational methodologies, shALIGN and shRNAseq, that allow RNAi screens to be rapidly deconvoluted using next generation sequencing. Our computational pipeline offers efficient screen analysis and the flexibility and scalability to quickly incorporate future developments in shRNA library technology.