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Second-generation shRNA libraries covering the mouse and human genomes

Nature Genetics volume 37, pages 1281–1288 (2005)Cite this article

Abstract

Loss-of-function phenotypes often hold the key to understanding the connections and biological functions of biochemical pathways. We and others previously constructed libraries of short hairpin RNAs that allow systematic analysis of RNA interference–induced phenotypes in mammalian cells. Here we report the construction and validation of second-generation short hairpin RNA expression libraries designed using an increased knowledge of RNA interference biochemistry. These constructs include silencing triggers designed to mimic a natural microRNA primary transcript, and each target sequence was selected on the basis of thermodynamic criteria for optimal small RNA performance. Biochemical and phenotypic assays indicate that the new libraries are substantially improved over first-generation reagents. We generated large-scale-arrayed, sequence-verified libraries comprising more than 140,000 second-generation short hairpin RNA expression plasmids, covering a substantial fraction of all predicted genes in the human and mouse genomes. These libraries are available to the scientific community.

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Figure 1: Design and structure of shRNA-mir cassettes.
Figure 2: Construction of the second-generation library.
Figure 3: Validation of the second-generation library.
Figure 4: Performance of the second-generation library in a small-scale high-throughput screen.

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Acknowledgements

We thank members of the laboratories of G.J.H., S.J.E. and S.W. Lowe for suggestions; J. Magnus for assistance with array PCRs; L. Nascimento, V. Balija, M. Kramer, T. Zutavern, S. Muller and B. Miller for assistance with sequencing; T. Moore for help with curation of the collection; and P. Linsley and S. Friend for their support of this project. This work was funded in part by awards from the Department of Defense Breast Cancer Research Program (G.J.H. and S.J.E.) and the US National Institutes of Health (G.J.H. and S.J.E.). S.J.E. and G.J.H. are investigators of the Howard Hughes Medical Institute.

Author information

Author notes

  1. Jose M Silva, Mamie Z Li and Ken Chang: These authors contributed equally to this work.

Authors and Affiliations

  1. Cold Spring Harbor Laboratory, Watson School of Biological Sciences, 1 Bungtown Road, Cold Spring Harbor, 11724, New York, USA

    Jose M Silva, Ken Chang, Michael C Golding, Despina Siolas, Patrick J Paddison, Nihar Sheth, Ravi Sachidanandam, W Richard McCombie & Gregory J Hannon

  2. Department of Genetics, Center for Genetics and Genomics, Brigham and Women's Hospital, Harvard Medical School, Room 158D, NRB, 77 Avenue Louis Pasteur, Boston, 02115, Massachusetts, USA

    Mamie Z Li, Richard J Rickles, Guang Hu, Michael R Schlabach & Stephen J Elledge

  3. Rosetta Inpharmatics LLC, 401 Terry Ave. North, Seattle, 98109, Washington, USA

    Wei Ge, Jeff Bradshaw, Julia Burchard, Amit Kulkarni, Guy Cavet & Michele A Cleary

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  1. Jose M Silva
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Corresponding authors

Correspondence to Stephen J Elledge or Gregory J Hannon.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Mapping of Dicer and Drosha cleavage sites. (PDF 16 kb)

Supplementary Fig. 2

The complete insert sequences for pSM1 and pSM2 containing a luciferase shRNA are shown along with their most stable potential secondary structures as predicted by RNA fold. (PDF 100 kb)

Supplementary Fig. 3

Stable suppression by pSM2. (PDF 212 kb)

Supplementary Table 1

ShRNAs used in Figure 3. (PDF 86 kb)

Supplementary Table 2

ShRNAs used in Figure 4. (PDF 41 kb)

Supplementary Table 3

Oligonucleotides used in construction of the library vectors. (PDF 17 kb)

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Silva, J., Li, M., Chang, K. et al. Second-generation shRNA libraries covering the mouse and human genomes. Nat Genet 37, 1281–1288 (2005). https://doi.org/10.1038/ng1650

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