Pooled screening overview

In pooled shRNA screening, hundreds to thousands of hairpins are combined (pooled) and interrogated simultaneously in a multiplex assay without the need for robotics or liquid handling.
The schematic to the below depicts this process.

• 1. Transduction: Cells are transduced at a low MOI ensuring a single shRNA is expressed in each cell.
• 2. Screen: The screen employs a selection process that is specific to the researcher’s assay.  There are two underlying strategies for selection: Negative selection (dropout) screens include an untreated control for comparison to allow the detection of shRNA that provided resistance or sensitized the cells to the selective reagent. Positive selection (enrichment) screens only detect surviving cells and do not require an untreated control.
• 3. Analysis: Under selection, resistant cells increase in the population and sensitized cells decrease in the population.  These changes in representation can be detected by sequence analysis (either Sanger sequencing or Next Generation Sequencing).

Pooled screening assays

The underlying workflow of all pooled shRNA screens begins by transducing cells with a heterogeneous pool of viral particles followed by an assay that enriches specific cells based on a phenotypic change.  Many variations have been successfully used in vivoor in vitro. However, most fall into three categories:  viability screen, reporter screen or behavioral screen1,2

Viability Screen:

In viability screens, the pool is most often used in combination  with a selective agent (e.g. drug treatment, exposure to pathogens).  An shRNA’s impact on cell health is measured by changes in its representation in population.  A negative impact on cell viability will decrease an shRNA’s representation while a positive impact on cell viability will increase representation3,4,5.

Reporter Screens:

Reporters, such as fluorescent markers, can be used to indicate changes in transcription or can be fused to proteins to measure stability or localization.  Cells can be sorted for high or low levels of expression and hairpins from each group can be analyzed for their enrichment or depletion8.

Behavioral Screens:

Cell behavior can also be assessed in a pooled screening assay.  For example, colony formation on soft agar can be used to select for cells with anchorage independent growth which can be an indicator of oncogenic transformation9,10.

In addition, one of the advantages of shRNA over siRNA is the ability to perform these screens in vivo as well as in vitro.  Viability screens have been successfully adapted using xenographs to identify tumor suppressor genes as well as oncogenes5,6,7.

This range of application in addition to the accessibility of shRNA pools provides researchers with a powerful option for screening.

Documents

Technical manual

shERWOOD-Ultramir Lentiviral shRNA Pooled Library – Plasmid
shERWOOD-Ultramir Lentiviral shRNA Pooled Library – Viral Particles
ZIP Promoter Selection Kit

References

1.  Sims D. et al.  (2011) High-throughput RNA interference screening using pooled shRNA libraries and next generation sequencing. Genome Biology, 12(10):R104
2. Hu G and Luo J. (2012) A primer on using pooled shRNA libraries for functional genomic screens.  Acta Biochim Biophys Sin. Feb;44(2):103-12
3. Westerman B. et al. (2011) A genome-wide RNAi screen in mouse embryonic stem cells identifies Mp1 as a key mediator of differentiation. J Exp Med. Dec 19; 208(13): 2675–2689
4. Schlabach M. et al. (2008) Cancer Proliferation Gene Discovery Through Functional Genomics Science 1 February 2008: Vol. 319 no. 5863 pp. 620-624
5. Luo, J. et al.  (2009) A Genome-wide RNAi Screen Identifies Multiple Synthetic Lethal Interactions with the Ras Oncogene. Cell Volume 137, Issue 5, p835–848, 29 May
6. Wuestefeld, T. et al. (2013) A Direct In Vivo RNAi Screen Identifies MKK4 as a Key Regulator of Liver Regeneration.  Cell Apr 11; 153(2): 389–401
7. Possemato, R. et al. (2011) Functional genomics reveal that the serine synthesis pathway is essential in breast cancer. Nature Aug 18; 476(7360): 346–350
8. Zender, L. et al. (2008) An Oncogenomics-based in vivo RNAi screen identifies tumor suppressors in liver cancer. Cell. Nov 28; 135(5): 852–864
9. Gazin , C. et al. (2007) An Elaborate Pathway Required for Ras-Mediated Epigenetic Silencing. Nature, Oct 25; 449(7165): 1073-1077
10. Smolen G. et al. (2010) A genome-wide RNAi screen identifies multiple RSK-dependent regulators of cell migration. Genes Dev ;24:v2654-2665.
11. Westbrook T. et al. (2005) A genetic screen for candidate tumor suppressors identifies REST. Cell. 121(6): 837–848.

Choice of pomoter,reporter, gene content and more

Achieve superior RNAi screens with enhanced potency, sequence-verified  shRNA pools and flexible delivery strategies. 

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 

Custom Pooled shRNA Libraries

Custom pools can be created from any shRNA content, 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@skyangbio.com for more information and pricing

Pooled screening deconvolution

Pooled shRNA sequencing and deconvolution service 

Need help deconvoluting your pooled screen? Send us your genomic DNA samples for sequencing and analysis.

• Simplify your screen – Sequencing service including library preparation, next generation sequencing and data analysis
• Cost effective – Multiplex sequencing allows analysis of multiple samples in parallel
• Quick turnaround – 4-6 weeks from receipt of samples to delivery of data
• More data reproducibility – Optimized protocols and reagents for shRNA specific sequencing

In addition to providing the highest quality sensor-based shRNA pooled library, we also offer next generation sequencing analysis and deconvolution services. Provide us with the genomic DNA from the samples following your screen and we will provide the precise number of sequencing reads of each individual clone within a pooled shRNA sample.

Pricing is based on the number of samples and the number of shRNAs in a pool.
Please contact us on info@skyangbio.com for further information and pricing.