Number of harvested cells as a baseline for human SAM Screen?

I am performing genome-wide screening with SAM library.
I have a question about the required number of cells for amplification of gDNA as control of baseline transduction at 3DPI - "S. Konermann et al., Nature 2014".

In this paper it is said that >500 cells per guide were harvested as a baseline 3DPI although 3DPI means 2 days of selection which is not time enough to have a 100% selection with the antibiotic.

Therefore harvesting just 500 x 70,290 (#of sgRNAs) cells at 3DPI would not be a real 500x coverage since there is still a % of non-transduced alive cells by that time - Am I wrong?


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Fuchsia Kelpiealmost 5 years ago

I believe more cells were harvested at 3DPI to account for incomplete antibiotic selection. We recently published a protocol associated with SAM screening that you might find helpful.

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