Second-generation shRNA libraries covering the mouse and human genomes.
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.
Silva
Jose M
JM
Cold Spring Harbor Laboratory, Watson School of Biological Sciences, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.
Li
Mamie Z
MZ
Chang
Ken
K
Ge
Wei
W
Golding
Michael C
MC
Rickles
Richard J
RJ
Siolas
Despina
D
Hu
Guang
G
Paddison
Patrick J
PJ
Schlabach
Michael R
MR
Sheth
Nihar
N
Bradshaw
Jeff
J
Burchard
Julia
J
Kulkarni
Amit
A
Cavet
Guy
G
Sachidanandam
Ravi
R
McCombie
W Richard
WR
Cleary
Michele A
MA
Elledge
Stephen J
SJ
Hannon
Gregory J
GJ
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*
D035683:MicroRNAs; D034741:RNA, Small Interfering