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.
Genomic DNA (gDNA) from the screen is isolated from the samples of interest. While performing the screen, 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. An overview of the provided data files is described in
Screen Deconvolution with PoolQ.
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.
