Timepoints for NGS/Sequencing of Screening Cells with GecKO V2

today the question comes up at which timepoints NGS of the library transduced cells are recommended, during the screening experiment.
Normaly i would say,

1. sequencing the amplified libarary plasmid.
2. after transduction and 7 days of puromycin Selection = d0 Start of screening experiment
3. after finishing your screen with the substance of choice =dx end of experiment.

The question is, would it make sense to sequence also after transduction and before selection with Puromycin?


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Fuchsia Kelpiealmost 5 years ago

For NGS time points, I would definitely sequence the amplified plasmid for QC and at the end of the experiment (treated and control samples). Typically, I would sequence 2 days after transduction of the library (when cells have been selected for 2 days with Puro) if I am looking for essential genes and need an early time point to which I can normalize the guide distribution.

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