Pooled shRNA Libraries

VectorBuilder offers high-quality, premade pooled shRNA libraries targeting human and mouse genes. These RNAi libraries can serve as powerful and cost-efficient tools for performing large-scale loss-of-function screens for genes involved in disease pathways, cell responses to drug treatment, developmental processes, gene regulation, etc. We can also design and construct custom pooled shRNA libraries based on your target gene list. 

Types of pooled shRNA libraries offered
  • Ready-to-use, pooled, lentivirus libraries targeting human genes at two scales: Elite Gene (~2,000 most frequently cited genes on PubMed Central) and Whole Genome (~19,000 RefSeq genes)
  • Ready-to-use, pooled, lentivirus libraries targeting mouse genes at two scales: Elite Gene (~2,000 most frequently cited genes on PubMed Central) and Whole Genome (~19,000 RefSeq genes)
  • Custom pooled shRNA libraries (available as E.coli stock, DNA or recombinant virus)

Ordering Information

Price and turnaround
Product Name No. of genes No. of shRNAs Scale Catalog No. Price (USD) Turnaround
Human Elite Gene Pooled shRNA Library 2,161 12,471

Medium

(>1.0x108 TU/ml, 1 ml)

LVM(Lib190505-1037bjk) $4,999
$2,499
10-20 days
Mouse Elite Gene Pooled shRNA Library 2,233 12,472

Medium

(>1.0x108 TU/ml, 1 ml)

LVM(Lib190505-1039sgb) $4,999
$2,499
Human Whole Genome Pooled shRNA Library 18,432 92,917

Medium

(>1.0x108 TU/ml, 1 ml)

LVM(Lib190505-1038ngq) $4,999
$2,499

Plus

(>1.0x108 TU/ml, 5 ml)

LV5M(Lib190505-1038ngq) $14,999
$7,499
Mouse Whole Genome Pooled shRNA Library 19,790 92,917

Medium

(>1.0x108 TU/ml, 1 ml)

LVM(Lib190505-1042tfx) $4,999
$2,499

Plus

(>1.0x108 TU/ml, 5 ml)

LV5M(Lib190505-1042tfx) $14,999
$7,499

Click to view our custom library construction services if our premade shRNA libraries don’t meet your needs 

Deconvolution service
Not sure how to identify shRNA hits by NGS after conducting your shRNA library 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.
For deconvolution service, please Send Design Request to us.

Technical Information

Product highlights View more

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 (~2,000 most frequently cited genes on 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. Detailed lists of target genes and shRNAs can be found under “Technical documents”.

High uniformity

The pooled plasmid libraries have been validated by next-generation sequencing (NGS), which has successfully detected 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 plasmid libraries (Figure 1).

Figure 1. Representation of shRNAs in different pooled plasmid 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 2. Map of mammalian shRNA knockdown lentiviral vector.

Click to view fully annotated map and sequence of the shRNA library vector 

Click to read more about 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 3. EGFP expression in 293T cells transduced with Human Elite Gene Pooled shRNA Library (MOI=10) after 4 days of puromycin selection (1.5 ug/ml). Magnification: 200x. Left: bright field. Right: EGFP.

Workflow of pooled shRNA library-mediated genetic screens View more

A conventional workflow of genetic screens mediated by pooled shRNA lentivirus libraries is shown in Figure 4 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(s) 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 4. Workflow of loss-of-function screens mediated by pooled shRNA lentivirus libraries. Adapted from Acta Biochim Biophys Sin 44:103-112 (2012).

FAQ

What are the advantages of pooled RNAi screen compared to array-based RNAi screen?

Pooled RNAi screen has a number of advantages compared to array-based RNAi screen, which are listed in the table below:

Array-based Screen Pooled RNAi 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).
Technical variation High Low
Cost of labor and reagents High Low
Requirement of special equipment High (e.g. liquid handler, high-throughput imaging, etc.) Low (conventional benchtop equipment)
How is shRNA knockdown score calculated?

VectorBuilder applies rules similar to that used by the RNAi consortium (TRC) to design and score shRNAs. For each given RefSeq transcript, we search for all possible 21mers that are considered as candidate target sites. Candidates are excluded if they contain features thought to reduce knockdown efficiency/specificity or cloneability, including a run of ≥4 of the same base, a run of ≥7 G or C, GC content <25% or >60%, and AA at the 5’ end. Knockdown scores are penalized for candidates that contain internal stem-loop, high GC content toward the 3’ end, known miRNA seed sequences, or off-target matches to other genes. For genes with alternative transcripts, target sites that exist in all transcripts are given higher scores.

All scores are ≥0, with mean at ~5, standard deviation at ~5, and 95% of scores ≤15. An shRNA with a knockdown score about 15 is considered to have the best knockdown performance and cloneability, while an shRNA with a knockdown score of 0 has the worst knockdown performance or is hard to be cloned.

Please note that knockdown scores are only a rough guide. Actual knockdown efficiency could depart significantly from what the scores predict. Target sites with low scores may still work well. Also, please note that targeting 3’ UTR can be as effective as targeting coding region.