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Custom Pooled Lentiviral Screening Libraries

Design the ideal pooled library for your experimental needs

When a Dharmacon catalog screening library does not meet your needs, we can help you design your own custom lentiviral pools. Select from a range of our algorithm-optimized reagents to develop the ideal screening resource for CRISPR-Cas9 knockout or RNAi knockdown screens.

Features

  • Pools of lentiviral sgRNA, shRNA, or microRNA
  • Customizable promoter and reporter options, including inducible promoters
  • Complete workflow planning tools and protocols
  • Human, mouse, and rat libraries available

Custom lentiviral pooled screening libraries offer a personalized and efficient method for screening large numbers of lentiviral sgRNAs, shRNAs, or microRNAs without the requirement of automation. When a Dharmacon catalog screening library does not meet your needs, we can help you design your own custom lentiviral pools. All you need to do it selecting the product format desired, the number of genes, constructs per gene, and species.

Optimized construction of high-quality pools, complete analysis tools, and validated protocols are essential to a successful outcome when screening with pooled lentiviral libraries. Whether you need to perform a CRISPR knockout screen with lentiviral sgRNAs, a knockdown RNAi screen with shRNA, or a gene regulation screen with expressed microRNAs, a custom library can be built to your experimental requirements.

Library construction, pooling techniques, and high-throughput sequencing compatible screening workflows have all been experimentally validated to ensure reproducibility and accurate hit identification. Pools are provided as concentrated lentiviral particles for transduction into dividing and non-dividing cells.

Experimentally validated protocols and calculation sheets are readily available to help you with every aspect of the experiment from determining relative titer in your specific cells to calculating exactly how much volume you will require to complete the experiment. Our Bioinformatic analysis protocol provides full instructions on using open source software to run the analysis from the NGS data.

To learn more about the critical parameters of successful pooled lentiviral screening, including the conditions necessary for maintaining a high 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).

Specifications:

Titer: Constitutive vectors, 5 x 108 TU/mL (± 20%); inducible vectors, 1 x 107 TU/mL (± 20%); all products are purified lentiviral particles with functional titers determined by qPCR. Pool size: minimum of 50 total constructs up to 12,000 constructs per pool Volume: Minimum of 100 uL up to 5 mL per pool; delivered in 25 µL aliquots

Custom lentiviral pooled libraries can be generated from any of the following product lines:

Edit-R Lentiviral sgRNA Number of targeted genes (protein-coding only)Average designs per geneAvailable controls*Compatible NGS Analysis kit
Human 185259.8100 NTC
340 positive
Edit-R Pooled sgRNA Indexing PCR and Sequencing Primer Kits
Mouse 196839.8100 NTC
340 positive
Edit-R Pooled sgRNA Indexing PCR and Sequencing Primer Kits
SMARTvector Lentiviral shRNA Number of targeted genes (includes lncRNA genes)Average designs per geneAvailable controls*Compatible NGS Analysis kit
Human 192419.5100 NTC
272 positive
SMARTvector Indexing PCR and Sequencing Primer Kits
Mouse 217459.2100 NTC
272 positive
SMARTvector Indexing PCR and Sequencing Primer Kits
Rat 226469.2100 NTC
272 positive
SMARTvector Indexing PCR and Sequencing Primer Kits
shMIMIC Lentiviral microRNA Number of mature microRNAsNumber of unique designsAvailable Controls*Compatible NGS Analysis kit
Human 2580255530 NTC
80 positive
SMARTvector Indexing PCR and Sequencing Primer Kits
Mouse 1913189630 NTC
80 positive SMARTvector
Indexing PCR and Sequencing Primer Kits
GIPZ Lentiviral shRNA (Decode) Number of targeted genesAverage designs per geneAvailable controls*Compatible NGS Analysis kit
Human 182055.3 1 NTC
2 positive
Decode Indexing PCR and Sequencing Primer Kit
Mouse180455.61 NTC
2 positive
Decode Indexing PCR and Sequencing Primer Kit

High quality pooled screening begins with rigorous library production

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

Successful pooled screening requires a high construct fold representation

Critical to the success of your screen and identification of quality hits is performing the screen at a high fold representation (the extent to which any given construct (e.g., shRNA) in a pooled library will be represented in the screen). High construct 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 representation after phenotypic selection.

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

  • All constitutive promoter lentiviral screening libraries are provided as concentrated (≥ 108TU/mL) lentiviral particles in sufficient quantity to transduce multiple biological replicates. Libraries with an inducible reporter are provided at ≥ 107TU/mL.
  • Optimized and experimentally validated product-specific PCR primers are designed to efficiently amplify genomic DNA with minimal bias and allow downstream high-throughput sequencing analysis of construct abundance
  • Sufficient quantity of lentiviral particles, PCR and sequencing primers allow the maintenance of high fold representation throughout the entire screening process

Successful pooled screening requires a high fold representation of each construct

Each step of the 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). Dharmacon pooled screening systems includes Illumina-adapted PCR primers for identification of hairpin sequences from gDNA by high-throughput sequencing on Illumina instrumentation. Vector-specific primer pairs are optimized for efficient amplification of the construct while minimizing thermodynamic bias and variation in representation. In addition, 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 hairpin insert facilitates data analysis and ensures accurate target gene identification.