HT RNAi TGF-β Screen Comparison Data
A comparison of the performance of a second generation validated tet-inducible 25K Druggable Genome shRNA library and two commercially-available non-validated shRNA libraries reveals that Cellecta has made significant improvements over other pooled library technology currently available. A genetic screen experiment performed in collaboration with Dr. Peiqing Sun at The Scripps Research Institute was designed to identify modulators that confer resistance to TGF-β-induced apoptosis in the Hep3B human hepatocellular carcinoma cell line.
Methods
Hep3B cells were transduced with a Cellecta 25K shRNA/6.5K target Validated library, Competitor #1 25K, and Competitor #2 200K human lentiviral shRNA libraries under similar conditions (MOI=0.5) with a 50 to 100-fold redundancy above the complexity of the library, seeded at low density, and treated with TGF-β (1 ng/ml) for three weeks. The shRNA inserts that block TGF-β apoptosis were amplified from genomic DNA of TGF-β-resistant cells and identified by conventional sequencing (Competitor library #1), hybridization with the Affymetrix U133+2 array (Competitor library #2), or HT sequencing using a Illumina-Solexa Genome Analyzer (Cellecta library). The comparative TGF-β genetic screens were performed in duplicate, and the set of identified known TGF-β effectors was compiled.
Results
For Competitor library #1, we were able to identify only TGF-β receptor type 1. For Competitor library #2, we identified only SMAD4 and another gene not previously implicated in TGF-β signaling. Moreover, the confirmation experiments with individual shRNA constructs identified in the primary screen with competitor shRNA libraries revealed a high (90-95%) rate of false positives. In contrast, the RNAi screen using Cellecta's Validated 25K shRNA library allowed us to identify more than 100 known and novel genes involved in TGF-β signaling, with high overlap between replicates (about 80%) and a high confirmation rate (about 95%). In addition to identifying a very high percentage of known TGF-β signaling pathway components (such as SMAD4, type I and type II TGF-β receptors, and WNT5A), the screen performed with the Validated shRNA library also revealed that the PI3K/NF-kB pathway plays an important role in TGF-β-mediated apoptosis of Hep3B cells, judging from the high number of pathway components identified by the screen.
Discussion
Overall, the high confirmation rate, overlap between replicates, and the high number of known TGF-β signaling pathway and general apoptosis genes identified from the Validated 25K library clearly indicate that this library has a much higher success rate than other commercially-available libraries when used in genetic screens. It is important to note that all the genes targeted by the Validated 25K library are also included in competitor library #2. The difference in the screening efficiency is thus not due to differential coverage of genes by these two libraries. Our results demonstrate that the combination of optimal shRNA design, the choice of functionally validated shRNAs, and the improved approach for shRNA hit identification based on Illumina-Solexa high-throughput bar-code sequencing have greatly increased the power of our Validated shRNA libraries over other currently available shRNA libraries.
View our Frequently Asked Questions (FAQs) page for detailed information on our pooled lentiviral shRNA libraries. For relevant references and literature, visit the Articles page under Resources. Visit the HT RNAi Viability Screen in DU145 for additional functional data.
Related Products & Services
Back to Top
