Designing and Building Effective Pooled shRNA Libraries
Pooled lentiviral-based libraries containing heterogeneous mixtures of shRNA constructs allow you to assay the effects of many thousands of pooled shRNA-expressing constructs in one experiment. Although Cellecta’s shRNA screening libraries are generated using standard, proven genetic library construction techniques, there are a number of technical challenges to produce quality shRNA expression libraries.
Quality Oligos
For large-scale production of heterogeneous populations of designed oligonucleotides for complex libraries, Cellecta has partnered with Agilent Technologies. Agilent’s microarray-based oligonucleotide synthesis platform is unparalleled in terms of providing full-length oligonucleotides over 100 bases in length with minimal mutations (mutation rates approximately 0.2%). Also, of critical importance, the solid support synthesis minimizes bias by providing similar levels of each individual species.
Well-Designed Vectors
To provide efficient delivery of complex shRNA libraries into different cell types for a variety of experimental designs, we have developed HIV-based lentiviral shRNA cloning vectors with H1 or U6 tet-regulated or constitutive promoters for expression of shRNA and a choice of a single or dual selection marker (GFP, RFP, PuroR, BleoR, etc.) expressed from a single CMV, EF1, PGK, UbiC, or other promoter. Cellecta's HIV-based lentivectors have abroad range of tropisms for efficient transduction in a wide variety of cells.
Unambiguous Sequenceable Bar-Codes
Quality control of the libraries and the final screening representation analysis is greatly facilitated by the incorporation of easily sequenced bar-codes in each shRNA construct. The bar-codes enable unambiguous identification of each shRNA species with HT sequencing.
Effective shRNA
In constructing an shRNA expression library, one key challenge is the design and production of quality oligonucleotides to provide the shRNA inserts for the libraries. Cellecta has developed its own in-house shRNA design algorithm that makes use of internal studies primarily focused mostly on structural features (e.g., length, loop size, mismatches, etc.), combined with published information regarding sequence preferences, including known sequences shown effective for a particular target.
Representation Levels of Individual shRNA Sequences
Cellecta specifically designs and constructs pooled shRNA libraries using proven library construction procedures, not by re-amplifying and mixing pre-made individual shRNA constructs. As a result, it is possible to obtain a very narrow representation of virtually all shRNA. The use of our “intelligent” bar-coding in combination with HT sequencing enables Cellecta to ensure that more than 99% of shRNA are present in every library. We typically achieve 80-90% representation within a 10-fold range.
Upper panel shows a library with very good representation. Virtually all the shRNA are present in more than 100 copies and most are around 1,000 copies. Also, there is less than 10-fold difference between the most represented and least for about 80-90% of the shRNA, so the library has a relatively balanced representation of all shRNA. The lower panel shows a poor library where almost half of the shRNA are present at less than 100 copies and the distribution is very broad. It is only possible to get readable signals for less half the targets using the the library in the lower panel.
This definitive representation data at the start of a screening provides a starting point for the analysis to find shRNA that significantly increase or decrease during screening, indicating relevant targets. Without this starting point, it is difficult to differentiate signal vs. noise in any screening assays—or even which shRNA is actually missing in the screen. In other words, you need this data to know what is truly being screened.
Quantifiable HT Sequencing
HT sequencing significantly outperforms the hybridization-based approach for identification of individual shRNA species based on the high-quality “digital expression data” generated by using bar-codes. Even using optimized bar-code sequences, array hybridization suffers from a limited dynamic range of approximately 2 orders of magnitude which results in a loss of as much as 30% of the signals that fall outside their effective range. Also, spot-to-spot cross hybridization on arrays results in significant noise that does not occur with HT sequencing where virtually every shRNA in the population is detected and counted, from those present in only a few copies to those present in several million. Differences in shRNA species between control and test populations are very easily detected and statistically analyzed, so that hits can be confidently identified.
Comprehensive
Data-Rich Results
Cellecta’s pooled libraries offer the capability of targeted screens knocking out the same transcripts with multiple shRNAs that serve to verify a specific functional effect and rule out off-target effects. In addition to unbiased wide-range screens of many thousands of genes to identify ones involved in a particular response, screens with small, highly-redundant targeted libraries provide invaluable approaches to thoroughly investigate the function of key pathways, analyze sets of known genes involved in specific responses, or validate targets identified in earlier large-scale screens.
View our Frequently Asked Questions (FAQs) page for detailed information on our pooled lentiviral shRNA libraries.
Review our custom shRNA library construction services.
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