Rapid creation and quantitative monitoring of high coverage shRNA libraries.
Short hairpin RNA libraries are limited by low efficacy of many shRNAs and by off-target effects, which give rise to false negatives and false positives, respectively. Here we present a strategy for rapidly creating expanded shRNA pools (approximately 30 shRNAs per gene) that are analyzed by deep sequencing (EXPAND). This approach enables identification of multiple effective target-specific shRNAs from a complex pool, allowing a rigorous statistical evaluation of true hits.
Bassik
Michael C
MC
Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biomedical Research, Howard Hughes Medical Institute, University of California, San Francisco, California, USA.
Lebbink
Robert Jan
RJ
Churchman
L Stirling
LS
Ingolia
Nicholas T
NT
Patena
Weronika
W
LeProust
Emily M
EM
Schuldiner
Maya
M
Weissman
Jonathan S
JS
McManus
Michael T
MT
D001483:Base Sequence; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics*; D017423:Sequence Analysis, RNA / Q000379:methods*
D012333:RNA, Messenger