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 (∼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.
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Acknowledgements
We would like to thank A. Brincat from the Sandler Lentiviral RNAi Core and C. McArthur from the Sandler Asthma Basic Research Center for technical assistance. We would also like to thank Q. Mitrovich and N. Goddard for technical advice, and D. Hirschberg and T. Baxter of Agilent Technologies. This work was supported by a Rubicon grant from The Netherlands Organization for Scientific Research (NWO) to R.J.L. and by a Career Development Fellowship from the Leukemia and Lymphoma Society to M.C.B. M.S. was supported by a postdoctoral fellowship from the Sandler Program in Basic Sciences and is now supported by an US National Institutes of Health K99/R00 (Pathway to Independence) award. N.T.I. is supported by the US National Institutes of Health under a Ruth L. Kirschstein National Research Service Award (GM080853) from the National Institute of General Medical Sciences. This work was supported by a Sandler New Technologies grant to J.S.W. and M.T.M., US National Institutes of Health grant RO1 GM80783 to M.T.M. and a grant from the Fight For Mike foundation to J.S.W.
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E.M.L. is employed by Agilent Technologies, Inc., and Agilent reagents are used in the research presented in this article.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–6, Supplementary Tables 1–4, Supplementary Notes 1 and 2 (PDF 1742 kb)
Supplementary Data 1
CD antigen shRNA library sequences (PDF 1242 kb)
Supplementary Data 2
CD45 shRNA sequences (PDF 27 kb)
Supplementary Data 3
LAIR1 shRNA sequences (PDF 18 kb)
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Bassik, M., Lebbink, R., Churchman, L. et al. Rapid creation and quantitative monitoring of high coverage shRNA libraries. Nat Methods 6, 443–445 (2009). https://doi.org/10.1038/nmeth.1330
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DOI: https://doi.org/10.1038/nmeth.1330
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