The basis of Cellecta’s RNAi genetic screening technology is the stable suppression of specific genes on a large-scale using pooled shRNA libraries, allowing for loss-of-function screens in mammalian cell systems. Genetic screens with shRNA libraries can be utilized to investigate most aspects of biology that can be recapitulated in a cell culture model. As opposed to expressing and assaying the functional effects of an individual shRNA molecule, the development of complex shRNA libraries allows for simultaneous screening of thousands of different shRNA molecules on a target population. In general, genetic screens represent an unbiased approach to identify genes that act in specific cellular pathways. HT RNAi genetic screens have been proven to be an extremely potent and versatile tool to explore the molecular basis of cancer development and progression, and to discover genes essential for viability in cancer cells that can be used as targets for anticancer drug development.
The screening process introduces a lentiviral library encoding a highly heterogeneous population of barcoded shRNA constructs that are packaged in viral particles pseudotyped with the vesicular stomatitis virus glycoprotein G (VSV-g) into a population of cells under conditions where most of the cells only take up and express a single gene-specific shRNA. On completion of the screen, cells exhibiting the desired phenotypic changes are isolated, and the shRNA constructs, presumably inducing the phenotypes, are recovered by PCR and identified by Next-Generation Sequencing (NGS) of shRNA-specific barcodes.
Major advantages of VSV-g pseudotyped lentivectors for shRNA library delivery are broad spectrum transduction tropism, allowing efficient transduction of non-transfectable cell types and tissues, long-term silencing of target genes, presenting the possibility of screening functions (senescence, differentiation, growth in soft agar, etc.) that require weeks rather than days in vitro or ex vivo.
Genetic screens using pooled shRNA libraries have the requirement for recipient cells with desired phenotypic changes to be selected from a pool of unaffected cells. Selection may be based on cell survival, appearance of specific markers, induction of reporter constructs, changes in cell morphology or behavior, etc. The design of a selection strategy is the most critical arm of any genetic screen project. Repeated rounds of selection may be necessary for either secondary validation or to reduce the number of false positives thereby increasing the percentage of positive hits.
Cellecta’s shRNA libraries also contain shRNA-specific barcodes. The barcodes can be read by NGS on the Illumina platform. Identified barcodes can be converted to lists of genes/shRNAs using our enumerated barcode data analysis software. The inclusion of clonal barcodes allows for identification of shRNAs without the need for amplification or sequencing of the hairpins themselves, which can be cumbersome due to the secondary structure present in them. For more information on how the clonal barcodes were built and how they are useful, please read: https://cellecta.com/blogs/news/millions-of-defined-sequenceable-barcodes-for-clonal-cell-tracking-2.
Custom Pooled shRNA Libraries
For Custom Pooled shRNA Libraries, the structure of the vectors, barcodes, and shRNA sequences are specified by the customer. These details are present on the Product Certificate (PC or PAC) that is distributed along with the Custom shRNA Library. Please refer to this Product Certificate as necessary. The protocols below provide the instructions on how to package the plasmid form of the shRNA library into viral particles and guidelines for the preparation of barcoded probes for NGS and analysis of raw sequencing data sets. Please read the entire user manual before proceeding with your experiment. For a description of the theories behind using pooled shRNA lentiviral libraries, information on transduction of target cells, viral targeting, or for examples of positive and negative screens using pooled lentiviral libraries, please refer to the Screening Recommendations section.
Human Genome-Wide Pooled Lentiviral shRNA Library
The Human Genome-Wide Pooled Lentiviral shRNA Library (hGW) targets virtually all (19,268) protein-encoding genes. It covers all genes in the human genome, and each gene is targeted by 8 hairpins. We have incorporated clonal barcodes to enable you to track growth, differentiation, or migration of specific cells containing a specific shRNA throughout your experiment. The hGW consists of three modules, each covering ~6,500 genes. Since each gene is targeted by 8 hairpins, there are a total of 50,000- 55,000 hairpins per module. The modules are made with non-overlapping barcodes so that they can be combined to form a complete genome-wide shRNA library. Each hairpin in the hGW includes a clonal barcode, which facilitates NGS data analysis, identification of functional shRNAs, and allows for tracking of specific shRNAs in individual cells. Each 55K hGW library module also includes a panel of internal controls. The control block consists of 2 shRNA sequences targeting PSMA1, 2 shRNA sequences targeting RPL30, and 4 shRNA sequences targeting Luciferase. Each shRNA sequence is replicated 5 times with 5 different barcodes. This 5-replicate internal control is useful for assessing internal noise, because the 5 replicates for each shRNA should elicit the same phenotype.
DECIPHER Pooled Lentiviral shRNA Libraries
The DECIPHER Pooled shRNA Libraries were Cellecta’s first pooled shRNA libraries. There are 3 human modules, and 2 mouse modules of these libraries. Each modules targets ca. 5,000 genes with 5-6 shRNA to gene target. They cover much of the human and mouse gene set, but are not completely genome-wide. The barcodes in the DECIPHER libraries are composed of an 18-nt sequence. Although the barcodes are unique to each shRNA within a module, there is some overlap between barcodes in different modules, so the modules should not be mixed for screening or sequencing.
References and Product Citations for all Cellecta products can be found on the Cellecta website:
https://cellecta.com/pages/citations-publications.
Please read the entire user manual before proceeding with your experiment. Also, please note that, when working with pseudoviral particles, you should follow the recommended guidelines for working with Biosafety Level 2 (BSL-2) materials.
PLEASE NOTE: The purchase of all Cellecta Products is covered by Cellecta’s standard Terms and Conditions of Sale as described on Cellecta’s website, and selected Products containing particular technology or having certain features are also subject to restrictions on use as outlined in the Label License section of our website. Please review these Terms and Label License Restrictions before opening and using your Product and, if you are not able to abide by the restriction, contact Cellecta to return the item to Cellecta for a full refund.
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