What is TRC shRNA?
The RNAi Consortium, or TRC, is a public-private effort based at the Broad whose mission is to create a shRNA library as well to validate tools and methods that will enable the scientific community to use RNAi to determine the function of human and mouse genes.
What is shRNA screening?
Screening with RNAi is a powerful method for perturbing gene activity in cultured cells to enable gene function, pathway analysis, and target identification. shRNA is utilized for stable gene knockdown and two formats are commonly used for shRNA screening: arrayed libraries and pooled libraries.
How were the TRC shRNA constructs designed?
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 to 5) constructs were created for each target gene.
What are the TRC lentiviral shRNA libraries made of?
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 (100 μg/mL). We check all cultures for growth prior to shipment.
How many Refseq transcript variants do shRNAs target?
As a general rule in the construction of the library, we construct shRNAs targeting just one Refseq transcript for each NCBI gene. Because of the high sequence identity among different transcripts from the same gene, the majority of the shRNAs target all known transcript variants.
How are candidate shRNAs selected?
Candidates are ranked by adjusted score and then assessed for target region and overlap with other existing or ordered shRNAs until the desired number of candidates is selected per gene.