Decode Pooled Lentiviral shRNA Libraries

Decode Pooled Lentiviral shRNA Libraries

Introduction

Screen hundreds or thousands of genes without the need for costly automation.

Decode Pooled Lentiviral shRNA Screening Libraries are pools of GIPZ shRNAs targeting human genes, provided as concentrated lentiviral particles. 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. In contrast to the costly automated techniques that are required to screen using individually arrayed RNAi reagents, Decode pooled shRNA screening libraries allow the researcher to transduce and screen a population of cells within a few tissue culture dishes.

To learn more about the critical parameters of successful pooled lentiviral shRNA screening, including the conditions necessary for maintaining a high shRNA fold-representation, please download the following publication: Ž. Strezoska, A. Licon, Optimized PCR Conditions and Increased shRNA Fold Representation Improve Reproducibility of Pooled shRNA Screens. PLoS One 7, e42341 (2012).

The Decode Pooled Lentiviral shRNA Screening platform includes:

  • High-quality shRNA pools provided as concentrated lentiviral particles and available as human gene family, druggable and whole genome libraries
  • Non-targeting control for transduction optimization
  • Optimized and 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
  • Experimentally tested protocols and planning tools for successful pooled lentiviral RNAi screens

Available Decode Pooled Lentiviral shRNA Screening Libraries

Library Catalog Number Number of genes targeted Number of pools x number of shRNA constructs per pool Lentiviral particle volume per pool
Ubiquitin Conjugate RHS6076 571 1 pool of 3,830 shRNA 2 tubes x 25 µL (50 µL total)
Phosphatase RHS6077 254 1 pool of 1,561 shRNA 2 tubes x 25 µL (50 µL total)
Protein Kinase RHS6078 709 1 pool of 4,675 shRNA 2 tubes x 25 µL (50 µL total)
Ion Channel RHS6079 374 1 pool of 1,884 shRNA 2 tubes x 25 µL (50 µL total)
GPCR RHS6080 382 1 pool of 2,591 shRNA 2 tubes x 25 µL (50 µL total)
Protease RHS6081 478 1 pool of 2,559 shRNA 2 tubes x 25 µL (50 µL total)
Druggable Genome RHS6082 7494 5 pools of 8,490 shRNA 4 tubes x 25 µL (100 µL total)
Human Genome RHS6083 18205 10 pools of 9,570 shRNA 4 tubes x 25 µL (100 µL total)



Decode pooled shRNA screening protocols begin by transducing cells at a low multiplicity of infection (MOI) with a lentiviral pool containing between 1,500 and 10,000 unique shRNAs. Following transduction, a selective pressure is applied to identify shRNAs that target genes involved in a specific biological response. As a result of the selective pressure, cells expressing shRNA are either enriched or depleted in the cellular population. To identify hits, genomic DNA is isolated from the initial transduced cell population (reference cells) and from the transduced cell population that remains following the application of selective pressure and phenotypic selection (experimental cells). The differences in shRNA abundance between reference and experimental cell populations can then be determined.

Decode Pooled shRNA Library Screening Workflow


The Decode Pooled Lentiviral shRNA Screening Libraries combine the advantages of the microRNA-adapted GIPZ shRNA design with the convenience of high-titer lentiviral delivery to create a powerful multiplexed RNAi screening resource capable of producing loss-of-function phenotypes in many dividing and non-dividing cells.

Phusion Products Accuracy Chart 1

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.



GIPZ shRNA Vector and Silencing Performance


pGIPZ

Vector Element Utility
hCMV Promoter Human cytomegalovirus promoter drives strong transgene expression
tGFP TurboGFP reporter for visual tracking of transduction and expression
PuroR Puromycin resistance permits antibiotic-selective pressure and propogation of stable integrants
IRES Internal ribosomal entry site allows expression of TurboGPF and puromycin resistance genes in a single transcript
shRNA microRNA-adapted shRNA (based on miR-30) for gene knockdown
5' LTR 5' long terminal repeat
3' SIN-LTR 3' self-inactivating long terminal repeat for increased lentivirus safety
ψ Psi packaging sequence allows viral genome packaging using lentiviral packaging systems
RRE Rev response element enhances titer by increasing packaging efficiency of full-length viral genomes
WPRE Woodchuck hepatitis post-transcriptional regulatory element enhances transgene expression in the target cells


pGIPZ

GIPZ shRNAs effectively knockdown genes of interest. HEK293T cells were transduced with lentiviral particles containing shRNA to the indicated genes and a non-silencing (NS) control at an average MOI of 10. 48 hours post- transduction, cells were selected with puromycin for four days prior to RNA isolation. Expression of indicated genes was determined by RT-qPCR and normalized to non-silencing control.

Pooled shRNA Screening

Achieve reliable and reproducible RNAi screening hits with the Decode pooled shRNA screening platform through:

  • stringently prepared shRNA pools with QC by next-generation sequencing (NGS)
  • empirically validated, comprehensive protocols for optimization, screening and hit analysis by high-throughput sequencing
  • enough concentrated lentiviral particles to perform well-designed screens at high shRNA fold representation

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.

Successful pooled shRNA screening starts with high-quality shRNA pools

Decode shRNA pools are created using experimentally validated methods that ensure uniform representation of shRNA constructs in every lentiviral pool. After pooling GIPZ shRNAs, DNA are prepared from E. coli cultures and analyzed by high-throughput sequencing to evaluate shRNA representation and identity. This quality control allows Dharmacon to verify that > 95% of shRNAs are recovered after the pooling process and the abundance of 70% of the shRNAs is less than 5-fold different from each other and the abundance of 90% of the shRNAs is less than 25-fold different from each other. This quality control provides confidence in the uniformity of the Decode pooled RNAi screening libraries and the ability to detect changes in shRNA representation.

Decoded PCR amplification Chart

Plasmid DNA was isolated from the Decode Phosphatase glycerol stock pool containing 1561 shRNAs targeting 254 genes (A). Genomic DNA was isolated from HEK293T cells that were transduced with the Decode Phosphatase library at a 1000-fold shRNA representation (B). Both DNA samples were sequenced on an Illumina Hi-Seq to determine the shRNA represented in the samples. Histograms of the normalized log2 counts show that shRNAs recovered after the pooling process (glycerol stock) and after transduction with lentiviral particles are represented with tight distributions. This ensures there is ample representation of each shRNA in the screening experiment.

shRNA Fold Representation

Successful pooled shRNA screening requires a high shRNA fold representation

Critical to the success of your screen and identification of quality hits is performing the screen at a high shRNA fold representation (the extent to which any given shRNA construct in a pooled library will be represented in the screen). High shRNA representation results in a greater degree of reproducibility between biological replicates and ensures that there is a sufficient experimental window for detection of changes in shRNAs representation after phenotypic selection.

Dharmacon provides the tools necessary to reproducibly identify hits with confidence:

  • Decode screening libraries are provided as concentrated (≥ 108 TU/mL) lentiviral particles in sufficient quantity to transduce multiple biological replicates
  • Optimized and experimentally validated PCR primers are designed to efficiently amplify genomic DNA with minimal bias and allow downstream high-throughput sequencing analysis of shRNA abundance
  • Sufficient quantity of lentiviral particles, PCR and sequencing primers allow the maintenance of high shRNA fold representation throughout the screening process

Receiver Operating Characteristic Chart

The Receiver Operating Characteristic (ROC) curves for a screen of 10,000 shRNAs at 100- (red) and 500-fold (blue) shRNA representation for detecting two-fold enrichment and depletion. The 500-fold screen has a larger area under the ROC curve and a higher true positive rate than the 100-fold screen, demonstrating the superior ability of the 500-fold screen to detect enriched and depleted shRNAs.

Genomic DNA PCR and High-throughput Sequencing

Successful pooled shRNA screening requires a high shRNA fold representation

Each step of the Decode pooled screening workflow, from transduction to hit identification, has been empirically tested. Amplification conditions were identified to ensure uniform amplification and high reproducibility (Strezoska et al. 2012). The Decode pooled shRNA screening system includes Illumina-adapted PCR primers for identification of shRNA hairpin sequences from gDNA by high-throughput sequencing on Illumina instrumentation. Decode primer pairs are optimized for efficient amplification of shRNA while minimizing thermodynamic bias and variation in representation. In addition, Decode PCR primers have built-in adaptor and index sequences that allow the researcher to easily move from PCR amplification to Illumina high-throughput sequencing. Direct identification of shRNA passenger strand facilitates data analysis and ensures accurate target gene identification.

pGIPZ

PCR amplification and Illumina high-throughput sequencing workflow.

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. 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 GIPZ lentiviral shRNAs.



Next-generation sequencing protocol is reproducible so that experimental noise is reduced and hits are more easily identified.

The Decode Phosphatase pooled screening library was transduced into HEK293T cells at 1000-fold shRNA representation. Cells were selected with puromycin for 72 hours and genomic DNA (gDNA) was isolated. Decode PCR and Sequencing primers were used to amplify and sequence gDNA samples as two technical duplicates (blue) and two different transductions (red) and counts analyzed by Illumina high-throughput sequencing. The log2 counts of shRNAs for each of the replicates are plotted against each other.

Technical replicate 2 log Chart

Reproducibility between replicates is maintained through PCR steps

Reproducibility between replicates is maintained through PCR steps by following Decode experimental protocols.

The Decode phosphatase library was transduced into HEK293T cells at 1,000-fold representation. Cells were selected with puromycin for 72 hours and genomic (gDNA) was isolated. Decode PCR primers were used to amplify shRNA sequences at indicated fold representations. Technical replicates of each PCR were compared to determine reproducibility (Pearson Correlation), where 1.0 indicates that the samples are 100% identical.

PCR template amount and cycle number