Decode Pooled Lentiviral shRNA Screening Libraries and Reagents

Screen hundreds or thousands of genes in dividing and non-dividing cells without the need for costly automation.


Decode Pooled Lentiviral shRNA Screening Libraries and Reagents
Through RNAi-mediated silencing of hundreds or thousands of genes in parallel, a pooled lentiviral shRNA screen can be performed to identify genes that regulate cellular responses and signaling pathways, or to discover novel gene functions within a few tissue culture dishes.
Decode lentiviral shRNA screening libraries offer an efficient and cost-effective method for screening large numbers of shRNAs without automation. Decode library construction, pooling techniques, and high-throughput sequencing compatible screening workflows have been experimentally validated to ensure reproducibility and accurate hit identification. Decode libraries are offered for a variety of targeted gene families as well as the human druggable genome and whole genome to fit many experimental designs.

Decode lentiviral RNAi screening libraries are pools of GIPZ short hairpin RNAs (shRNAs) that target human genes provided as concentrated lentiviral particles for transduction into dividing and non-dividing cells. Through RNAi-mediated silencing of hundreds or thousands of genes in parallel, a pooled lentiviral shRNA screen can be performed to identify genes that regulate cellular responses and signaling pathways, or to discover novel gene functions within a few tissue culture dishes. Decode pooled screening libraries contain an average of five shRNAs per target gene and are supplied in sufficient volume of lentiviral particles to allow high biological reproducibility in relevant cells that are refractory to transfection. Also available for use with the Decode screening libraries are optimized PCR and sequencing primers, high fidelity DNA polymerase and experimentally tested protocols for reliable identification of shRNA hits by high-throughput sequencing.

Custom Decode Pooled Lentiviral shRNA Screening Libraires targeting human or mouse genes are available upon request. Request pricing for a custom pooled library.

 

Available Decode Pooled Lentiviral shRNA Screening Libraries
Library Catalog # # of genes targeted Number of pools x number of shRNAs per pool Lentiviral particle volume per pool
Ubiquitin Conjugation RHS6076 571 1 pool of 3,830 shRNA 2 tubes × 25 μL (50 μL total)
Phosphatase RHS6077 254 1 pool of 1,561 shRNA 2 tubes × 25 μL (50 μL total)
Protein Kinase RHS6078 709 1 pool of 4,675 shRNA 2 tubes × 25 μL (50 μL total)
Ion Channel RHS6079 347 1 pool of 1,884 shRNA 2 tubes × 25 μL (50 μL total)
GPCR RHS6080 382 1 pool of 2,591 shRNA 2 tubes × 25 μL(50 μL total)
Protease RHS6081 478 1 pool of 2,559 shRNA 2 tubes × 25 μL(50 μL total)
Druggable Genome RHS6082 7,494 5 pools of 8,490 shRNA 4 tubes × 25 μL(100 μL total)
Human Genome RHS6083 18,205 10 pools of 9,570 shRNA 4 tubes × 25 μL(100 μL total)

Before ordering, download these invaluable tools to carefully plan your pooled lentiviral shRNA screen and calculate the amounts of components required:

Additional products recommended in the validated Decode screening protocol:

  • Non-targeting control for transduction optimization (Cat #RHS4348)
  • Decode Indexing PCR and Sequencing Primer Kit (Cat #PRM6178) which includes optimized and experimentally validated primers for
    • Efficient PCR amplification of genomic DNA with minimal bias
    • High-throughput multiplexed sequencing for hit identification
  • Phusion Hot Start II High Fidelity DNA Polymerase, the most accurate hot start DNA polymerase on the market (Cat #F-549S, F-549L)

Note: We recommend purchasing two Decode Indexing PCR and Sequencing Kits for use with the Decode Human Genome Library.

Important Notice

The Decode Pooled Lentiviral Screening Libraries are solely for internal research use (as set forth in the Product Terms and Conditions) in laboratories where the containment measures stated below and in applicable laws and regulations are met. Products may not be used for diagnostic, therapeutic or other commercial purposes and may not to be administered to humans for any purpose or to animals for therapeutic purposes.Decode Pooled Lentiviral Screening Librariesare provided as lentiviral particles are replication-incompetent, self-inactivating (SIN) and non-pathogenic (do not cause infectious human disease).

Any investigator who purchases lentiviral particle products is responsible for consulting with their institution's health and biosafety personnel for specific guidelines on the handling of lentiviral vector particles. Furthermore, each investigator is fully responsible for obtaining the required permissions for research using and the acceptance of replication-incompetent SIN lentiviral vectors and replication-defective lentiviral particles into their local jurisdiction and institution.

  
HazardousNo
Shipping ConditionDry Ice,Wet Ice
Stability at Recommended Storage ConditionsAt least 12 months
Storage Condition-80 C,-20 C
Viral Titer>5X108TU/mL
pGIPZ vector cartoon.jpg

pGIPZ vector cartoon

pGIPZ vector cartoon.jpg


GIPZ knockdown data graph.jpg

GIPZ knockdown data graph

GIPZ knockdown data graph.jpg

OVCAR-8 cells were transduced with GIPZ lentiviral shRNA constructs at MOI = 0.4 to2 in 2 to 4 biological replicates. Cells were puromycin-selected (30 μg/mL) starting 48 hours post-transduction. RNA was isolated 84 hours posttransduction. qPCR was performed in triplicate via TaqMan® Gene Expression Assays using 18S rRNA as an internal reference. On average, two out of three shRNA produced greater than 70% knockdown compared to the GIPZ Non-targeting Control shRNA.


Pooled-shRNA-screening-workflow

Pooled-shRNA-screening-workflow

Pooled-shRNA-screening-workflow

Pooled shRNA screening workflow. Assay Development and Optimization: Establish optimal experimental conditions, including those for a) lentiviral transduction and b) screening parameters, such as selective pressure and time between collection of reference and experimental samples. Primary Screen: A stable population of cells expressing single integrants of shRNAs are created by transducing Decode lentiviral pools at low MOIs. Transduced cells are then split into reference and experimental populations for application of a selective pressure that induces the phenotype of interest. Genomic DNA (gDNA) is then isolated from reference and experimental populations of transduced cells. Illumina-adapted primers and Phusion Hot-Start II High Fidelity DNA Polymerase are used to PCR amplify integrated shRNA sequences and add Illumina flow-cell binding sequences. The resulting amplicons are run on Illumina platform sequencers, using the sequencing primers provided. Hit Identification and Follow-up: shRNA sequences are identified in reference and experimental libraries. shRNAs that are enriched or depleted during the screen are identified as hits, and the genes that they target are identified. Hits can be confirmed and studied further using individual shRNA constructs that can be ordered from the GIPZ lentiviral shRNA collection.


References

  1. C. Gazin, N. Wajapeyee, An elaborate pathway required for Ras-mediated epigenetic silencing. Nature 449, 1073 (2007).
  2. S. Gobeil, X. Zhu, A genome-wide shRNA screen identifies GAS1 as a novel melanoma metastasis suppressor gene. Genes & Dev. 22, 2932 (2008).
  3. J. C. Kappes, X. Wu, Safety considerations in vector development. Somat. Cell Mol. Genet. 26, 147 (2001).
  4. B. L. Levine, L. M. Humeau, Gene transfer in humans using a conditionally replicating lentiviral vector. PNAS 103, 17372 (2006).
  5. M. R. Schlabach, J.Luo, Cancer Proliferation Gene Discovery Through Functional Genomics. Science 319, 620 (2008).
  6. J. M. Silva, K. Marran, Profiling essential genes in human mammary cells by multiplex RNAi screening. Science 319, 617 (2008).
  7. D. Sims, A. Mendes-Pereira, High-throughput RNA interference screening using pooled shRNA libraries and next generation sequencing. Genome Biol. 12, R104 (2011).

Citations

  1. Ž. Strezoska, A. Licon, Optimized PCR Conditions and Increased shRNA Fold Representation Improve Reproducibility of Pooled shRNA Screens. PLoS One 7, e42341 (2012).