OVCAR-8 cells were transduced with GIPZ lentiviral shRNA constructs at MOI = 0.4 to2 in 2 to 4 biological replicates. Cells were puromycin-selected (30 μg/mL) starting 48 hours post-transduction. RNA was isolated 84 hours posttransduction. qPCR was performed in triplicate via TaqMan® Gene Expression Assays using 18S rRNA as an internal reference. On average, two out of three shRNA produced greater than 70% knockdown compared to the GIPZ Non-targeting Control shRNA.
Pooled shRNA screening workflow. Assay Development and Optimization: Establish optimal experimental conditions, including those for a) lentiviral transduction and b) screening parameters, such as selective pressure and time between collection of reference and experimental samples. Primary Screen: A stable population of cells expressing single integrants of shRNAs are created by transducing Decode lentiviral pools at low MOIs. Transduced cells are then split into reference and experimental populations for application of a selective pressure that induces the phenotype of interest. Genomic DNA (gDNA) is then isolated from reference and experimental populations of transduced cells. Illumina-adapted primers and Phusion Hot-Start II High Fidelity DNA Polymerase are used to PCR amplify integrated shRNA sequences and add Illumina flow-cell binding sequences. The resulting amplicons are run on Illumina platform sequencers, using the sequencing primers provided. Hit Identification and Follow-up: shRNA sequences are identified in reference and experimental libraries. shRNAs that are enriched or depleted during the screen are identified as hits, and the genes that they target are identified. Hits can be confirmed and studied further using individual shRNA constructs that can be ordered from the GIPZ lentiviral shRNA collection.