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

• Published in: Genome biology Vol. 12; no. 10; pp. R104 - 2701
• Main Authors: Sims, David, Mendes-Pereira, Ana M, Frankum, Jessica, Burgess, Darren, Cerone, Maria-Antonietta, Lombardelli, Cristina, Mitsopoulos, Costas, Hakas, Jarle, Murugaesu, Nirupa, Isacke, Clare M, Fenwick, Kerry, Assiotis, Ioannis, Kozarewa, Iwanka, Zvelebil, Marketa, Ashworth, Alan, Lord, Christopher J
• Format: Journal Article
• Language: English
• Published: London Springer-Verlag 21.10.2011; BioMed Central
• Subjects: Algorithms; analysis; Animal Genetics and Genomics; Base Sequence; Bioinformatics; Biomedical and Life Sciences; Computational Biology; Computational Biology - methods; Evolutionary Biology; Gene Library; Genetic Vectors; Genetic Vectors - genetics; genetics; HEK293 Cells; Human Genetics; Humans; Lentivirus; Lentivirus - genetics; Life Sciences; Method; methods; Microbial Genetics and Genomics; phenotype; Plant Genetics and Genomics; Plasmids; Plasmids - genetics; Reproducibility of Results; RNA Interference; RNA, Small Interfering; RNA, Small Interfering - analysis; RNA, Small Interfering - genetics; screening; Sensitivity and Specificity; Sequence Alignment; Sequence Alignment - methods; Sequence Analysis, RNA; Sequence Analysis, RNA - methods; small interfering RNA; Transfection; User-Computer Interface; shRNA Sequence; Illumina GAIIx; shRNA Construct; Next Generation Sequencing; Viral Supernatant
• ISSN: 1465-6906; 1474-760X; 1465-6914; 1474-760X
• DOI: 10.1186/gb-2011-12-10-r104

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.