Rapid creation and quantitative monitoring of high coverage shRNA libraries (original) (raw)

Nature Methods volume 6, pages 443–445 (2009)Cite this article

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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Author notes

  1. Michael C Bassik and Robert Jan Lebbink: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biomedical Research, and Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California, USA
    Michael C Bassik, L Stirling Churchman, Nicholas T Ingolia, Weronika Patena, Maya Schuldiner & Jonathan S Weissman
  2. Department of Microbiology and Immunology, and University of California San Francisco Diabetes Center, University of California, San Francisco, San Francisco, California, USA
    Robert Jan Lebbink, Weronika Patena & Michael T McManus
  3. Genomics Solution Unit, Agilent Technologies Inc., Santa Clara, California, USA
    Emily M LeProust

Authors

  1. Michael C Bassik
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  2. Robert Jan Lebbink
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  3. L Stirling Churchman
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  4. Nicholas T Ingolia
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  5. Weronika Patena
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  6. Emily M LeProust
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  7. Maya Schuldiner
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  8. Jonathan S Weissman
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  9. Michael T McManus
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Corresponding authors

Correspondence toJonathan S Weissman or Michael T McManus.

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Competing interests

E.M.L. is employed by Agilent Technologies, Inc., and Agilent reagents are used in the research presented in this article.

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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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