title: Second-generation shRNA libraries covering the mouse and human genomes.
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.
authors.affiliation: Cold Spring Harbor Laboratory, Watson School of Biological Sciences, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.
authors.forename: Jose M
authors.initials: JM
authors.lastname: Silva
authors.forename: Mamie Z
authors.initials: MZ
authors.lastname: Li
authors.forename: Ken
authors.initials: K
authors.lastname: Chang
authors.forename: Wei
authors.initials: W
authors.lastname: Ge
authors.forename: Michael C
authors.initials: MC
authors.lastname: Golding
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Rickles
authors.forename: Despina
authors.initials: D
authors.lastname: Siolas
authors.forename: Guang
authors.initials: G
authors.lastname: Hu
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Paddison
authors.forename: Michael R
authors.initials: MR
authors.lastname: Schlabach
authors.forename: Nihar
authors.initials: N
authors.lastname: Sheth
authors.forename: Jeff
authors.initials: J
authors.lastname: Bradshaw
authors.forename: Julia
authors.initials: J
authors.lastname: Burchard
authors.forename: Amit
authors.initials: A
authors.lastname: Kulkarni
authors.forename: Guy
authors.initials: G
authors.lastname: Cavet
authors.forename: Ravi
authors.initials: R
authors.lastname: Sachidanandam
authors.forename: W Richard
authors.initials: WR
authors.lastname: McCombie
authors.forename: Michele A
authors.initials: MA
authors.lastname: Cleary
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Elledge
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Hannon
mesh_terms: D000818:Animals; D015723:Gene Library; D020868:Gene Silencing; D015894:Genome, Human; D006801:Humans; D051379:Mice / Q000235:genetics*; D035683:MicroRNAs / Q000378:metabolism; D010957:Plasmids; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*
chemical_list: D035683:MicroRNAs; D034741:RNA, Small Interfering