high-throughput rna interference screening using pooled shrna libraries and next generation sequencing使用混合成分高通量rna干扰筛选库和下一代测序.pdfVIP

Sims et al. Genome Biology 2011, 12:R104 /2011/12/10/R104 METHOD Open Access 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 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 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. Background The experimental use of synthetic siRNAs and shRNA- RNA interference (RNAi) facilitates the assessment of expressing plasmids has profoundly changed the way in gene function by silencing gene expression using syn- which loss of function experiments can be performed. Pre- thetic anti-sense oligonucleotides or plasmids. It exploits viously, techniques that were either more time consuming a physiological mechanism that represses gene expres-