Nat Methods. 2009 Jun;6(6):443-5. doi: 10.1038/nmeth.1330. Epub 2009 May 17.

Rapid creation and quantitative monitoring of high coverage shRNA libraries.

Bassik MC¹, Lebbink RJ, Churchman LS, Ingolia NT, Patena W, LeProust EM, Schuldiner M, Weissman JS, McManus MT.

Author information

1: Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biomedical Research, Howard Hughes Medical Institute, University of California, San Francisco, California, USA.

Abstract

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.