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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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.
The authors declare no competing financial interests.
Mapping of Dicer and Drosha cleavage sites. (PDF 16 kb)
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)
Stable suppression by pSM2. (PDF 212 kb)
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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