Technical question: genomic screen with suspension cells

I’m planning a genomic screening with Jurkat cells (suspension cells), followed by treatment with a drug that should induce massive cell death.

I’m interested on the survivors that became resistant to the treatment, but I’m not sure if cleaning-up the dead cells (for example, with Ficoll), before NGS, is an essential step or not (since every cleaning step comes with the loss of some live cells).

So far, I can’t find a protocol that clearly has directions for that – anyone with experience in suspension cells in this context? Would you recommend to clean-up the dead cells at some point during my positive selective screen?

I would appreciate any suggestions and insights.

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Rose Valkyriealmost 5 years ago

We are trying a similar approach with AML cell lines as well. The problem with this methodology is that the number of surviving cells is a very small fraction and Ficoll/FACS sorting/MACS dead cell separation is unable to isolate such a small proportion of cells. Also, we have tried sorting necrotic cells from a drug-treated population using Annexin V/7-AAD sorting and found that they can also form a band during PCR2. Please let us know if there is any other way to perform a similar screen.

Pink Gremlinover 1 year ago

did you guys figure out how to remove the dead cell before genomic DNA extraction? I'm having the similar problem with my screening expt. Also, do you have any experience about the effect of dead cell contamination will cause?

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