Screening
When planning out the details of the screen, remember to think critically about maintaining library coverage at each step - library transduction, passaging, gDNA isolation, and sequencing. We recommend reviewing Coverage Considerations In Pooled Genetic Screens in detail.
Once the screen is done, genomic DNA (gDNA) is isolated from the samples of interest. It is highly recommended to perform a PCR efficiency test on a small quantity of gDNA from the screen, or, if the gDNA yield is on the lower end, on mock cells. Both steps are outlined in the Genomic DNA Isolation and PCR Pre-Check protocol.
After confirming successful amplification with the pre-check, the 96-well plates of samples can be submitted to GPP for PCR amplification and subsequent sequencing. The gDNA Submissions Portal contains all relevant information on this workflow. Once the samples have been sequenced, GPP will send a link to the deconvoluted data for analysis. GPP maintains a long-term back-up copy of the sequencing data (just in case).
Receipt of sequencing data is an excellent time to schedule to meet with a screening scientist.
FAQs:
Q: What is the protocol GPP uses to perform PCR on screening samples in 96-well plate format?
While we recommend having GPP perform the PCR and sequencing submission, as we are set up to do so in a high-throughput and cost-effective manner, our PCR of sgRNAs/shRNAs/ORFs from gDNA for Illumina Sequencing protocol is available for those who want to perform these steps on their own.