shRNA design and cloning:
The TRC shRNA constructs were designed using an siRNA rules based algorithm consisting of sequence, specificity and position scoring for optimal hairpins. The hairpin consists of a 21 base stem and a 6 base loop. The hairpins were cloned into the pLKO1 vector and sequence verified.
Multiple (4-5) constructs were created for each target gene. The pLKO1 lentiviral vector enables efficient transduction of primary and non-dividing cells such as neuronal cells making it easy to perform RNAi studies in these hard to transfect cell lines. Stable selection is also possible using the puromycin selectable marker.
Viral packaging:
Replication-incompetent viral particles can be efficiently produced using lentiviral packaging plasmids co-transfected in 293T packaging cells. The
Trans-Lentiviral packaging system which offers maximum biosafety and high titers is recommended.
ControlsAvailable
controls include the pLKO.1 empty vector as a negative control and an eGFP shRNA as a positive control.
Citing the library in Scientific Publications
In scientific publications, the libraries should be referred to as TRC-Hs1.0 (Human) and TRC-Mm1.0 (Mouse). Individual clones are uniquely identified by their TRC ID number (eg. TRCN0000014783).
Further information is available at http://www.broad.mit.edu/genome_bio/trc/rnai.html
The TRC lentiviral shRNA libraries are provided in 96-well microtiter plates containing frozen stock cultures of E. coli in 2XLB broth with 8% glycerol and carbenicillin (100ug/ml). Open Biosystems checks all cultures for growth prior to shipment. Individual shRNA constructs are shipped as glycerol stock cultures on wet ice. Store at -80ºC.
