DECIPHER Pooled Lentiviral shRNA Libraries
DECIPHER libraries are barcoded lentiviral shRNA libraries optimized for RNAi Genetic Screens in pooled format. They are made to cover the entire annotated human gene set and most annotated mouse gene set. Lentiviral shRNA libraries were chosen over synthetic siRNA collections for their advantages in cancer research, specifically for the ability to efficiently transduce a range of cell types, including non-dividing cells, and for the highly effective long-term gene silencing. The pooled format screens follow a simple protocol and do not require access to expensive high-throughput arrayed screen equipment and infrastructure, putting the genome-wide shRNA knockdown screens within reach of any researcher.
Designed Specifically for Screens in Pooled Format
Several DECIPHER library design features make them considerably more effective than other currently available RNAi libraries. The high percentage of functionally validated sequences and inclusion of a redundant set of 5 to 6 shRNA per gene results in at least 70% knockdown efficiency for approximately 65% of the target genes represented (depending on the cell type). A detailed description of one of our methods of choosing functional shRNAs is available on the Reporter Assay technology page.
The vast majority of shRNA constructs in the library are represented equally, with the difference in concentrations not exceeding one order of magnitude. Typically, about 80-90% of the population of shRNA constructs are present within a 10-fold range.
Intelligent barcode Design, Quality Data
Specially-designed, optimized 18-nt barcodes facilitate HT sequencing data analysis and identification of functional shRNAs. Using the Illumina HT Sequencing platform, barcodes are identified and converted to lists of genes/shRNA with enumerated barcode data. Amplification of shRNA hairpins is not required, as only the barcode sequence is required for identification. This enables unbiased amplification and unambiguous identification of each shRNA species.
The histograms above show the distribution of shRNA/barcode sequences in the DECIPHER plasmid shRNA library. Virtually all of the shRNA are present in more than 100 copies and most are around 1,000 copies per 20 million HT sequencing "reads". Also, there is less than a 10-fold difference between the most and least represented for about 80-90% of the shRNAs, so the libraries have a relatively balanced representation of all shRNAs.
Modular Library Format for Effective Screens
DECIPHER libraries are constructed in modules, with each module having 27,500 sequences and covering approximately 5000 genes. This allows to tailor the scope of the screen to a particular application and also circumvents the difficulty of performing negative selection (drop-out) viability screens, which are impractical for libraries over 30,000 constructs.
Modules 1 and 2 (Human and Mouse)
Two human and two mouse DECIPHER modules are included in the first release. The first module is intended for the widest possible screening applications. It consists of the following gene groups:
- Members of the major canonical and non-canonical pathways, covered by KEGG, Reactome, and other expert-curated pathway databases
- Genes on the CSHL Cancer 1000 list and 2500 top-ranking genes from the Cancer Genome Atlas, with priority based on the involvement in canonical pathways and disease association
- Known FDA drug targets
- Most important genes involved in top MeSH ranking diseases; these are chosen by their position within the pathway and number of nodes of connectivity
Thus, Module 1 is both versatile and wide in scope. However, Module 2 can be used for the more comprehensive follow-up screens or simply be done in parallel. There are approximately 10,000 genes that have an Entrez/RefSeq ID and at least one interaction documented in PubMed and cover the majority of disease-associated genes and drug targets. About half of these are included in Module 1 while Module 2 covers the rest.
Module 3 (Human)
Human Module 3, released in February 2011, targets 4,922 genes. This new Module adds additional cell surface, extracellular, DNA binding targets, and other genes to the well-annotated signal transduction and disease-associated genes primarily targeted with the first two modules of the human shRNA library. Many of the genes targeted in Module 3 are conserved and appear integral to a variety of important biological process but are not as well characterized as the 10,000 genes targeted in the first two modules.
The table below summarizes the DECIPHER Libraries currently available. For detailed information, including gene lists with complete annotations, user manual, and vector map/sequence, visit the Support page.
|DECIPHER 27K Pooled shRNA Library Modules||# of target mRNAs||shRNA complexity||Target Gene List|
|Human Module 1, Signaling Pathway Targets||5,043||27,500||Excel (XLS)|
|Human Module 2, Disease-Associated Targets||5,412||27,500||Excel (XLS)|
|Human Module 3, Cell Surface, Extracellular, and DNA Binding Targets||4,922||27,500||Excel (XLS)|
|Mouse Module 1, Signaling Pathway Targets||4,625||27,500||Excel (XLS)|
|Mouse Module 2, Disease-Associated Targets||4,520||27,500||Excel (XLS)|
NOTE: The module names are used solely for convenience to describe the major groups of genes targeted in the module. Many genes targeted in a module do not fall within the description, all modules target a variety of genes throughout the genome, and not all genes generally considered to fall under a specific description will be found in the module with the specific gene description. Please refer to the complete gene list associated with each module for detailed information regarding which genes are present in each specific module. Also, each module targets an orthogonal set of genes so there is no overlap in the targets between modules.
What you will receive in your order:
- 120 μg of each plasmid library ordered, in pRSI12-U6-(sh)-UbiC-TagRFP-2A-Puro vector; enough to generate virus for approximately 50-100 screens
- 10 μg empty library vector, for cloning individual constructs used to validate hits from your screen.
The Cellecta shRNA Library User Manual and Product Analysis Certificates (PACs) are now provided as PDF files and are available for download on the Support page.
Workflow of Library Preparation and RNAi Screening
Please read the Cellecta shRNA Library User Manual before starting, as careful planning is essential. The following briefly outlines the experimental workflow.
- Library packaging
- Titer estimation
- Transduction and screening
- DNA extraction
- Barcode amplification
- Data analysis and interpretation
DECIPHER Library Licensing
Licensing restrictions and use of the DECIPHER Libraries is covered by a Material Transfer Agreement (MTA). All researchers must agree to the MTA terms in order to obtain these library modules. Text of the Limited Use Label Licenses is included in the MTA. All IP generated from use of DECIPHER libraries belongs to the Recipient. For complete license and acceptable usage information, please refer to the DECIPHER MTA and NIH Data Sharing policy.
Please visit the Cellecta website for additional information on Pooled shRNA Libraries, RNAi Screening, and related custom services offered.