How to best pool multiple samples?

I'm doing a FACS based CRISPR screen. Due to the large physical size of the cells, I am unable to sort extremely large numbers of cells at once.

To get an adequate number of cells for good coverage, I'm doing multiple sorts/experiments that I would like to pool. However, I'm worried about biasing the library if one experiment is under/over represented in the pooled product.

So far, I've got:

Exp1: One sample was sorted into population A and B:

Population A: ~16M cells ->150 ug of gDNA

Population B : ~16M cells -> 110 ug of gDNA

Exp2 : One sample was sorted into population A and B:

Population A: ~15M cells -> 110 ug of gDNA

Population B : ~15M cells -> 100 ug of gDNA

So,

1. I'll do a few more passes for more cells.

2. Then I could pool all the "A" together for adequate coverage.

3. Then I'll sequence and compare "A" vs "B"

However, perhaps:

Exp1's "A" is over-represented in the pooled "A"

Exp2's "B" is over-represented in the pooled "B"

I don't think that would be very good. Exp1 and Exp2 probably had different initial guide biases in their pre-sorted populations, which would confound my results.

What would be the best way to pool the samples? Or should I perhaps never pool them, and instead use different Illumina adaptors for both Exp1 and Exp2 (and the remaining experiments)?


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Fuchsia Kelpieabout 4 years ago

I would suggest using different reverse primers (with different barcodes) for your experiments for now, and you can look at the data both separately and pooled to see which one looks better at the end. Whether you should pool the samples for the data analysis depends on your experiment, but if you use different reverse primers you can see both options.

Khaki Cerberusabout 4 years ago

Thank you!

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