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Cloned shRNA, knockdown guaranteed (Lentiviral Vector), Pooled Libraries

Pooled shRNA Screening Libraries
Screen thousands of shRNA in a single cell culture plate, without the need for robotics, with pooled shRNA screening libraries from transOMIC technologies. Pooled shRNA screening requires low multiplicity of infection (MOI) transductions to ensure that each cell in the population only expresses a single shRNA. This requires shRNA that are potent even at single integrations in the genome. transOMIC pooled shRNA screening libraries are created using shERWOOD algorithm-designed hairpins and are provided in a microRNA scaffold, which minimizes off-target effects (see link below).
Developed by Dr. Greg Hannon and colleagues at Cold Spring Harbor Laboratory, the algorithm selects designs based on those sequence biases and requirements shown by shRNA scored as potent in a pooled screen. This makes the new shERWOOD-based shRNA uniquely suited for pooled screening.

Pooled shRNA lentiviral libraries from transOMIC are produced using a proven process limiting variation in representation between shRNA constructs.


Choice of promoter, reporter, gene content and more:

Achieve superior RNAi screens with enhanced potency, sequence-verified shRNA pools and flexible delivery strategies. Gain greater confidence in your results.

• Every clone is sequence verified – eliminate unwanted background from mutations introduced in chip-based pools
• More potent shRNA per gene – decrease false positive and false negative results
• Equimolar pooling process - reduces variation between samples


Choose the best promoter for your cells

• Select the optimal promoter for your target cell line with a choice of promoters- choose from SFFV, hCMV, mCMV, hEF1a, and mEF1a


Your choice of Format

• In vitro or in vivo pool formats. Pools can be subdivided into minipools for lower complexity and higher representation for in vivo or other applications that require less complex pools
• Choice of gene coverage - premade gene families or custom gene sets
• Ready to use lentiviral particles or plasmid DNA format

Pool Deconvolution Service

• NGS analysis service - send us your genomic DNA and receive counts for every shRNA construct
• Optimized indexed primer kit available for NGS (Illumina platform)


Effective pooled screening is dependent on potent shRNA and equimolar representation

Image
Figure 1. Efficient single copy knockdown. Graph of expression level measured by western blot analysis showing representative knockdown in cell lines expressing shRNA from a single integration in the genome.

Pooled shRNA screening requires shRNA capable of knocking down its intended target while expressed from a single integration in the genome. Early generation shRNA libraries include a high number of shRNA constructs unable to achieve this possibly due to their designs being based on siRNA design rules rather than shRNA (which go through processing in the microRNA biogenesis pathway and have different requirements for optimal processing and function). In addition, shRNA pools created via chip-based method are known to contain a high number of mutations which limits their functionality. The shERWOOD algorithm efficiently selects designs that are potent at single copy (Figure 1) making them uniquely suited for pooled screening.
To learn more about the Sensor Screen underlying the shERWOOD algorithm see link below.



Image
Figure 2. Representative histogram for pooled shRNA library representation showing that >95% of constructs are within 5-fold of the mean.

Importance of equimolar distribution in a pooled shRNA screening library:
Changes in the relative distribution of the shRNA in a pool indicate a selective advantage or disadvantage during the screening process and are detected using next-generation sequencing (NGS). To ensure that artificial changes in population are not introduced, pools should be constructed to minimize variation in representation across hairpins. For example, false negative results can occur when an shRNA is under represented in the population and lost by chance due to low representation during transduction, insufficient cell plating during passaging, or insufficient sample size.
transOMIC pooled shRNA libraries start with sequence-verified arrayed shRNA resulting in better normalization of shRNA representation in pools. Typical pooled libraries have a relatively narrow range of shRNA distribution across the pool. >95% of the shRNA are within a 5-fold of the mean (Figure 2).



Choice of promoter for hard to transduce cells

Image

Figure 3. pZIP vectors options for pooled shRNA libraries. The fluorescent marker, puromycin resistance gene and shRNA are expressed from a single transcript making fluorescence a convenient and direct indicator of shRNA expression.

Mammalian promoters may differ in expression level or be silenced over time depending on the target cell line. Variation in expression level can affect fluorescent marker expression as well as knockdown efficiency. The Spleen Focus Forming Virus (SFFV) LTR provides a functional promoter in a number of human and mouse cell lines and is standard in the ZIP lentiviral vector used to create pooled shRNA screening libraries. However, for cell lines where a different promoter may be optimal, the ZIP promoter testing kit (TLN0005-TR) is available with a panel of vectors to quickly test for optimal expression in target cell lines. The ZIP lentiviral vector is available with five different promoter options (SFFV, human or murine CMV, human or mouse EF1-alpha) driving expression of shRNA (see schematic above). The fluorescent marker and shRNA are on the same transcript allowing the level of fluorescence in the cell to be used as a direct indication of shRNA expression through visual inspection. Ready-to-use viral particles for each of the five vectors are provided for transduction in target cell lines of interest to determine the optimal promoter for efficient shRNA expression.

The ZIP promoter selection kits (TLN0005-GVO-TR and TLN0005R-GVO-TR) provide sufficient viral particles of a non-targeting shRNA in each vector to transduce the target cell line and determine the optimal promoter.


Flexible pool formats for optimal pooled shRNA screens:

In vitro, in vivo and ex vivo screening strategies have different requirements for pool formatting. In vivo and ex vivo screens require smaller, less complex pools to facilitate a higher shRNA representation while in vitro screens can be efficiently performed with higher complexity pools. Pooled shRNA screening libraries are available as viral particles that are ready-to-screen or plasmid DNA that can be packaged as needed for small or large-scale screens. In addition pools have been subdivided into mini pools of ~1000 shRNA per pool to enable low complexity and higher representation for in vivo and other screening applications.


Screening capacity:

• Lentiviral pooled shRNA libraries provided as viral particles include enough virus to perform a screen at 500-1000-fold representation with at least 2 biological replicates.
• Libraries provided as plasmid DNA include enough plasmid for more than 3 packaging reactions. Total viral particles generated will depend on packaging efficiency, but is expected to provide enough viral particles to perform a screen in multiple cell lines at 500-1000-fold representation with 3 biological replicates.


Custom pools, deconvolution service and multiplex sequencing kits


Custom Pooled shRNA Libraries

Custom pools can be created from any shRNA Content you like, you can obtain plasmid DNA or lentiviral particles at titers up to 10^9TU/ml, formatted for in vitro, or in vivo screening, with a choice of promoters or fluorescent reporters. Contact us at info@biocat.com for more information and pricing.


Illumina Next Generation Sequencing Kit for Multiplex Analysis

NGS Sequencing kits have been optimized to allow multiple samples to be analyzed simultaneously saving time and sequencing costs. Index up to 12 samples for NGS multiplex analysis (Cat.# TRP0002-TR).


Deconvolution Services

A cost effective way to simplify your pooled RNAi screen Analysis: Send us the genomic DNA from your screen and we will return counts for each hairpin. Our optimized protocols provide reproducibility and a quick turnaround time (4-6 weeks).

Related Links

Contact us for a customized shRNA-mir library
shERWOOD-UltramiR shRNA
Info about shERWOOD shRNA-mir Algorithm
Pooled Screening Overview
Next Generation Sequencing Service


PDF-Downloads - Will open in new browser window

A Computational Algorithm to predict shRNA Potency (2014)

 

 

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Description Cat# Size Price    
Human Apoptosis Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library - Plasmid DNA, targeting 864 genes TLHUD7507-TRI 50 ug 8222 € DETAILS   Add to Cart 
Human Apoptosis Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library - Viral Particles, targeting 864 genes TLHUV7607-GVO-TRI 2 x 25ul of 10^8TU/ml 6902 € DETAILS   Add to Cart 
Human Apoptosis Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library, formatted as minipools of 1000 shRNA- Plasmid DNA, targeting 864 genes TLHUD7557-TRI 5 x 50 ug 9744 € DETAILS   Add to Cart 
Human Apoptosis Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library, formatted as minipools of 1000 shRNA- Viral Particles, targeting 864 genes TLHUV7657-GVO-TRI 5 x 2 x 25ul of 10^8TU/ml 8425 € DETAILS   Add to Cart 
Human B Cell Activation Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library - Plasmid DNA, targeting 102 genes TLHUD7521-TRI 50 ug 6699 € DETAILS   Add to Cart 
Human B Cell Activation Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library - Viral Particles, targeting 102 genes TLHUV7621-GVO-TRI 2 x 25ul of 10^8TU/ml 5380 € DETAILS   Add to Cart 
Human Breast Cancer Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library - Plasmid DNA, targeting 1049 genes TLHUD7527-TRI 50 ug 8222 € DETAILS   Add to Cart 
Human Breast Cancer Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library - Viral Particles, targeting 1049 genes TLHUV7627-GVO-TRI 2 x 25ul of 10^8TU/ml 6902 € DETAILS   Add to Cart 
Human Breast Cancer Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library, formatted as minipools of 1000 shRNA- Plasmid DNA, targeting 1049 genes TLHUD7577-TRI 5 x 50 ug 9744 € DETAILS   Add to Cart 
Human Breast Cancer Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library, formatted as minipools of 1000 shRNA- Viral Particles, targeting 1049 genes TLHUV7677-GVO-TRI 5 x 2 x 25ul of 10^8TU/ml 8425 € DETAILS   Add to Cart 
Human Breast Cancer specific Angiogenesis Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library - Plasmid DNA, targeting 42 genes TLHUD7531-TRI 50 ug 5177 € DETAILS   Add to Cart 
Human Breast Cancer specific Angiogenesis Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library - Viral Particles, targeting 42 genes TLHUV7631-GVO-TRI 2 x 25ul of 10^8TU/ml 3857 € DETAILS   Add to Cart 
Human Breast Cancer specific Metastasis Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library - Plasmid DNA, targeting 147 genes TLHUD7528-TRI 50 ug 6699 € DETAILS   Add to Cart 
Human Breast Cancer specific Metastasis Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library - Viral Particles, targeting 147 genes TLHUV7628-GVO-TRI 2 x 25ul of 10^8TU/ml 5380 € DETAILS   Add to Cart 
Human Bromodomain-containing Proteins Lentiviral ZIP (SFFV) Pooled shERWOOD Ultramir shRNA-mir library - Plasmid DNA, represents 507 clones targeting 43 genes TLH7035-TRI 50 ug please inquire € DETAILS   Add to Cart 
Human Bromodomain-containing Proteins Lentiviral ZIP (SFFV) Pooled shERWOOD Ultramir shRNA-mir library - Viral Particles, represents 507 clones targeting 43 genes TLH7135-GVO-TRI 2 x 25ul of 10^8TU/ml please inquire € DETAILS   Add to Cart 
Human Cancer Metabolism Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library - Plasmid DNA, represents 2187 clones targeting 642 genes TLHUD7532-TRI 50 ug 8222 € DETAILS   Add to Cart 
Human Cancer Metabolism Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library - Viral Particles, represents 2187 clones targeting 642 genes TLHUV7632-GVO-TRI 2 x 25ul of 10^8TU/ml 6902 € DETAILS   Add to Cart 
Human Cancer Metabolism Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library, formatted as minipools of 1000 shRNA- Plasmid DNA, represents 2187 clones targeting 642 genes TLHUD7548-TRI 2 x 50 ug 8729 € DETAILS   Add to Cart 
Human Cancer Metabolism Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library - formatted as minipools of 1000 shRNA, Viral Particles, represents 2187 clones targeting 642 genes TLHUV7648-GVO-TRI 2 x 2 x 25ul of 10^8TU/ml 8932 € DETAILS   Add to Cart 
Human Cell Adhesion Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library - Plasmid DNA, targeting 754 genes TLHUD7525-TRI 50 ug 8222 € DETAILS   Add to Cart 
Human Cell Adhesion Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library - Viral Particles, targeting 754 genes TLHUV7625-GVO-TRI 2 x 25ul of 10^8TU/ml 8425 € DETAILS   Add to Cart 
Human Cell Adhesion Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library, formatted as minipools of 1000 shRNA- Plasmid DNA, targeting 754 genes TLHUD7575-TRI 2 x 50 ug 8171 € DETAILS   Add to Cart 
Human Cell Adhesion Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library, formatted as minipools of 1000 shRNA- Viral Particles, targeting 754 genes TLHUV7675-GVO-TRI 2 x 2 x 25ul of 10^8TU/ml 10404 € DETAILS   Add to Cart 
Human Cell Cycle Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library - Plasmid DNA, targeting 1113 genes TLHUD7511-TRI 50 ug 9237 € DETAILS   Add to Cart 
Human Cell Cycle Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library - Viral Particles, targeting 1113 genes TLHUV7611-GVO-TRI 2 x 25ul of 10^8TU/ml 8222 € DETAILS   Add to Cart 
Human Cell Cycle Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library, formatted as minipools of 1000 shRNA- Plasmid DNA, targeting 1113 genes TLHUD7561-TRI 3 x 50 ug 9744 € DETAILS   Add to Cart 
Human Cell Cycle Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library, formatted as minipools of 1000 shRNA- Viral Particles, targeting 1113 genes TLHUV7661-GVO-TRI 3 x 2 x 25ul of 10^8TU/ml 10455 € DETAILS   Add to Cart 
Human Cytokines Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library - Plasmid DNA, targeting 206 genes TLHUD7517-TRI 50 ug 6699 € DETAILS   Add to Cart 
Human Cytokines Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library - Viral Particles, targeting 206 genes TLHUV7617-GVO-TRI 2 x 25ul of 10^8TU/ml 5380 € DETAILS   Add to Cart 
Human Cytokine Receptors Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library - Plasmid DNA, targeting 79 genes TLHUD7518-TRI 50 ug 6699 € DETAILS   Add to Cart 
Human Cytokine Receptors Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library - Viral Particles, targeting 79 genes TLHUV7618-GVO-TRI 2 x 25ul of 10^8TU/ml 5380 € DETAILS   Add to Cart 
Human Cytoskeleton Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library - Plasmid DNA, targeting 1719 genes TLHUD7524-TRI 50 ug 10252 € DETAILS   Add to Cart 
Human Cytoskeleton Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library - Viral Particles, targeting 1719 genes TLHUV7624-GVO-TRI 2 x 25ul of 10^8TU/ml 8425 € DETAILS   Add to Cart 
Human Cytoskeleton Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library, formatted as minipools of 1000 shRNA- Plasmid DNA, targeting 1719 genes TLHUD7574-TRI 5 x 50 ug 9744 € DETAILS   Add to Cart 
Human Cytoskeleton Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library, formatted as minipools of 1000 shRNA- Viral Particles, targeting 1719 genes TLHUV7674-GVO-TRI 5 x 2 x 25ul of 10^8TU/ml 10404 € DETAILS   Add to Cart 
Human Deubiquitinases Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library - Plasmid DNA, targeting 46 genes TLHUD7516-TRI 50 ug 5177 € DETAILS   Add to Cart 
Human Deubiquitinases Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library - Viral Particles, targeting 46 genes TLHUV7616-GVO-TRI 2 x 25ul of 10^8TU/ml 3857 € DETAILS   Add to Cart 
Human DNA Damage Response Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library - Plasmid DNA, targeting 570 genes TLHUD7513-TRI 50 ug 6699 € DETAILS   Add to Cart 
Human DNA Damage Response Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library - Viral Particles, targeting 570 genes TLHUV7613-GVO-TRI 2 x 25ul of 10^8TU/ml 6902 € DETAILS   Add to Cart 
Human DNA Damage Response Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library, formatted as minipools of 1000 shRNA- Plasmid DNA, targeting 570 genes TLHUD7563-TRI 5 x 50 ug 8171 € DETAILS   Add to Cart 
Human DNA Damage Response Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library, formatted as minipools of 1000 shRNA- Viral Particles, targeting 570 genes TLHUV7663-GVO-TRI 5 x 2 x 25ul of 10^8TU/ml 10404 € DETAILS   Add to Cart 
Human Epigenetics Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library- Plasmid DNA, represents 4185 clones targeting 406 genes TLHUD7509-TRI 50 ug 6699 € DETAILS   Add to Cart 
Human Epigenetics Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library- Viral particles, represents 4185 clones targeting 406 genes TLHUV7609-GVO-TRI 2 x 25ul of 10^8TU/ml 5380 € DETAILS   Add to Cart 
Human Epigenetics Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library, formatted as minipools of 1000 shRNA- Plasmid DNA, represents 4185 clones targeting 406 genes TLHUD7559-TRI 4 x 50 ug 8171 € DETAILS   Add to Cart 
Human Epigenetics Lentiviral ZIP (mCMV) Pooled shERWOOD Ultramir shRNA-mir library - formatted as minipools of 1000 shRNA, Viral Particles, represents 4185 clones targeting 406 genes TLHUV7659-GVO-TRI 4 x 2 x 25ul of 10^8TU/ml 10404 € DETAILS   Add to Cart 
Human Epigenetics Erasers Consolidated Set Lentiviral ZIP (SFFV) Pooled shERWOOD Ultramir shRNA-mir Library- Plasmid DNA, represents 522 clones targeting 44 genes TLH7044-TRI 50 ug please inquire € DETAILS   Add to Cart 
Human Epigenetics Erasers Consolidated Set Lentiviral ZIP (SFFV) Pooled shERWOOD Ultramir shRNA-mir Library- Lentiviral Particles, represents 522 clones targeting 44 genes TLH7144-GVO-TRI 2 x 25ul of 10^8TU/ml please inquire € DETAILS   Add to Cart 
Human Epigenetics Readers Consolidated Set Lentiviral ZIP (SFFV) Pooled shERWOOD Ultramir shRNA-mir Library- Plasmid DNA, represents 1238 clones targeting 194 genes TLH7042-TRI 50 ug please inquire € DETAILS   Add to Cart 
Human Epigenetics Readers Consolidated Set Lentiviral ZIP (SFFV) Pooled shERWOOD Ultramir shRNA-mir Library- Lentiviral Particles, represents 1238 clones targeting 194 genes TLH7142-GVO-TRI 2 x 25ul of 10^8TU/ml please inquire € DETAILS   Add to Cart 
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