Assay Development
The majority of the work that goes into a screen occurs beforehand in the assay development phase. Several experiments must be conducted to ensure proper design and execution of the screen, and we recommend following the steps outlined below:
Click on each step to learn more
5. Optimizing conditions for library delivery and post-screen deconvolution
- This is important to ensure one integration per cell.
- Empirically test a range of virus volumes by performing the Pooled Screen Spinfection Viral Titration or Pooled Screen No-Spin Viral Titration protocols.
- Take Coverage Considerations In Pooled Genetic Screens into account at this stage to achieve the most accurate screening results.
- While optimizing transduction and screen conditions, this is the best time to also test and optimize the Genomic DNA Isolation and PCR Pre-Check that occur after the screened cells are harvested. Reserve a population of transduced cells to test DNA isolation protocol(s) and ensure the subsequent PCR yields only the expected barcode-containing product suitable for sequencing.
- To order a small aliquot of library virus to test in the cells of interest, review Ordering Pooled Libraries.
FAQs:
Q: Can individual reagents be ordered for an assay development experiment?
GPP maintains individual (arrayed) reagents that can be provided as bacterial streaks, plasmid DNA and/or lentivirus:
- sgRNAs
- Largely for CRISPR knockout
- shRNAs
- Our collection of RNAi reagents
- ORFs
- Human Open Reading Frames
Q: Can individual customized reagents be generated for a screen?
GPP can produce custom sgRNA or shRNA constructs in 96-well plate format.
For small numbers of constructs, our recommended protocol is: Cloning of sgRNA or shRNA Constructs. To then prep the constructs as a glycerol or DNA stock, review our shRNA/sgRNA/ORF Glycerol and Plasmid DNA Preparation protocol.
Q: Can library plasmid DNA be amplified to make lentivirus?
GPP can amplify existing libraries to create more pDNA and/or lentivirus, following this protocol: pDNA Library Amplification.
Q: What protocol does GPP use to make lentivirus?
We use the following protocols for lentivirus production in varying formats:
For experiments that require a more concentrated form of lentivirus, review our Lentiviral Concentration protocol. To determine lentiviral titer, we recommend performing our Functional Viral Titering Assay. To produce VPX viral-like particles to improve transduction efficiency in challenging cell models, follow our protocol: Production of VPX Viral-Like Particles.