High-throughput RNA interference screening using pooled shRNA libraries and next generation sequencing.
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.
The Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK.
David
D
Sims
Ana M
AM
Mendes-Pereira
Jessica
J
Frankum
Darren
D
Burgess
Maria-Antonietta
MA
Cerone
Cristina
C
Lombardelli
Costas
C
Mitsopoulos
Jarle
J
Hakas
Nirupa
N
Murugaesu
Clare M
CM
Isacke
Kerry
K
Fenwick
Ioannis
I
Assiotis
Iwanka
I
Kozarewa
Marketa
M
Zvelebil
Alan
A
Ashworth
Christopher J
CJ
Lord
D000465:Algorithms; D001483:Base Sequence; D019295:Computational Biology / Q000379:methods*; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000032:analysis / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016415:Sequence Alignment / Q000379:methods*; D017423:Sequence Analysis, RNA / Q000379:methods; D014162:Transfection; D014584:User-Computer Interface
D034741:RNA, Small Interfering