Pooled shRNA Libraries
VectorBuilder offers high-quality pooled shRNA libraries targeting human and mouse genes. For each species, we provide ready-to-use lentiviral libraries at two scales: Whole Genome (~19,000 RefSeq genes) and Elite Gene (~2000 most frequently cited genes in PubMed Central). Where possible, each gene is targeted by 5-6 different shRNAs. These libraries can serve as powerful and cost-efficient tools for performing genome-wide loss-of-function screens in mammalian cells.
A conventional workflow of genetic screens mediated by pooled shRNA lentivirus libraries is shown in Figure 1 below. First, cells of interest are transduced with the shRNA lentivirus library, and positively transduced cells are selected by the marker gene(s) carried on the lentiviral genome (e.g. drug-selection or fluorescence marker). Next, positively transduced cells are split into reference population and experimental population. Then, the experimental population is subjected to particular selective pressure (e.g. drug treatment or repeated passaging) to identify cells with the phenotype of interest. There are three major types of screening strategies: 1) viability screens that search for shRNAs enriched or depleted in surviving cells when exposed to selective pressure; 2) reporter screens that look for shRNAs enriched in cells associated with either high or low reporter expression (e.g. shRNAs targeting transcription factors that modulate reporter gene expression); 3) behavior screens that usually identify shRNAs affecting genes associated with cell invasion, migration, etc. After screening, both experimental and reference cells are harvested, and enriched or depleted shRNAs in the experimental group compared to the reference group are identified using sanger sequencing or next-generation sequencing. Candidate genes that are potentially targeted by the enriched or depleted shRNAs can be further investigated by downstream functional studies.
Figure 1. Workflow of loss-of-function screens mediated by pooled shRNA lentivirus libraries. Adapted from Acta Biochim Biophys Sin 44:103-112 (2012).
Genome-wide targeting and high complexity: For both human and mouse, we offer shRNA libraries at two scales: Whole Genome (~19,000 RefSeq genes) and Elite Gene (~2000 most frequently cited genes in PubMed Central). On average, every gene is targeted by 5-6 different shRNAs with good knockdown scores calculated based on a set of rules following the guideline of the RNAi consortium (TRC). This enables more reliable knockdown and more effective screening.
High uniformity:The pooled libraries were validated by next-generation sequencing (NGS), which successfully recovered 100% of shRNAs in the Elite Gene libraries and >97% of shRNAs in the Whole Genome libraries. In addition, shRNA representation is highly uniform in the libraries (see Figure 2).
Figure 2. Representation of shRNAs in different pooled libraries. shRNA read counts are normalized by NGS library size (10 million reads) and plotted in log2 scale.
Well-established lentiviral vector and high titer lentivirus that is ready to use: The pooled shRNA libraries are expressed in the third-generation lentiviral vector system driven by human U6 promoter, which is a highly efficient system for stably knocking down expression of target genes in a wide variety of cells. This lentiviral vector system is ideal for in vitro genetic screens since it can introduce shRNAs into cells permanently, efficiently and uniformly. All pooled shRNA libraries are provided as ready-to-use lentivirus with high functional titer (>108 TU/ml), which saves your time in virus packaging and titer measurement. The third-generation lentiviral vector system is optimized for improved biosafety given its incompetent self-replication feature.
Figure 3. Map of the lentiviral shRNA knockdown vector.
Dual-marker for efficient and versatile selection or tracking of positively transduced cells: A dual-marker expression cassette of EGFP and puromycin resistance gene (Puro) is expressed from the lentiviral vector, allowing for selection of positively-transduced cells by puromycin and visual tracking by green fluorescence.
Figure 4. EGFP expression in 293T cells transduced with Human Elite Gene Pooled shRNA Library after 4 days of puromycin selection (1.5 ug/ml). Magnification: 200x. Left: bright field. Right: GFP.
Pooled shRNA screen: Pooled RNAi screen has a number of advantages compared to array-based RNAi screen, which is listed in the table below:
|Array-based Screen||Pooled Screen|
|How are shRNAs introduced into cells of interest?||Individual shRNAs are applied to cells grown in different wells across multi-well plates (e.g. 96-well or 384-well).||Hundreds and thousands of different shRNAs are applied to the cell population simultaneously.|
|How to identify shRNAs associated with phenotypes of interest?||Wells showing phenotypes of interest are selected by examining phenotypes well-by-well. The identity of the shRNAs applied to individual wells are already known.||Cells with phenotypes of interest are selected from the population. shRNAs enriched/depleted in cells of interest are identified by sequencing.|
|Can genetic interactions be detected?||No (if a single shRNA is added to each well), or limited (if more than one shRNAs are added to each well).||Yes (cells may carry multiple randomly combined shRNAs).|
|Cost of labor and reagents||High||Low|
|Requirement of special equipment||High (e.g. liquid handler, high-throughput imaging, etc.)||Low (conventional benchtop equipment)|
|Product Name||No. of genes||No. of shRNAs||Scale *||Catalog No.||Price (USD)|
|Human Elite Gene Pooled shRNA Library||2,161||12,470||Medium (>1.0x108 TU/ml, 1 ml)||Lib-LV-shRNA-hE-M||$4,999||Add to cart|
|Mouse Elite Gene Pooled shRNA Library||2,233||12,471||Medium (>1.0x108 TU/ml, 1 ml)||Lib-LV-shRNA-mE-M||$4,999||Add to cart|
|Human Whole Genome Pooled shRNA Library||18,432||92,917||Medium (>1.0x108 TU/ml, 1 ml)||Lib-LV-shRNA-hW-M||$4,999||Add to cart|
|Plus (>1.0x108 TU/ml, 5 ml)||Lib-LV-shRNA-hW-P||$14,999||Add to cart|
|Mouse Whole Genome Pooled shRNA Library||19,790||92,917||Medium (>1.0x108 TU/ml, 1 ml)||Lib-LV-shRNA-mW-M||$4,999||Add to cart|
|Plus (>1.0x108 TU/ml, 5 ml)||Lib-LV-shRNA-mW-P||$14,999||Add to cart|
* For Elite Gene libraries, the medium scale is sufficient for >50 screens at 100x shRNA coverage. For Whole Genome libraries, the medium scale is sufficient for >8 screens at 100x shRNA coverage, and the plus scale is sufficient for >40 screens at 100x shRNA coverage.
Not sure how to identify shRNA hits by NGS after conducting your screen? You can simply send the genomic DNA of your samples to VectorBuilder, and we will prepare the NGS libraries, perform high-throughput sequencing, analyze the data, and deliver the accurate counts of shRNAs in each sample to you in just a few weeks.
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