GeCKO V2 library screen

[Ariel Stanhill]

Dear All

I am using the GeCKO human KO library for a screen in a 293 reporter cell line.

As I understand from the Joung et al Nature protocol paper in a given 100,000 GeCKO library at least 5x10^7 cells need to be obtained for genomic DNA extraction (see step 57 in the protocol).

If a screen is performed in a GFP reporter cell line into which the GeCKO library is infected, does one obtain genomic DNA from unsorted (5x10^7 cells) and compare them to the GFP sorted cells? If only 2% are positively sorted is this sufficient? or do i need to 50x the number of initial cells to be sorted in order to end up with 5x10^7 positive cells?

thank you 

Ariel 

[Julia Joung]

Hi Ariel,

Thank you for your interest in our work. For FACS screens, sorting out 2% positive cells of the 5x10^7 cell population should be sufficient, because you will be able to harvest sufficient genomic DNA for PCR. For the control, I will typically sort out 5-10% of the low GFP cells rather than using the unsorted cells for better signal-to-noise.

Best,

Julia

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[Rehab Heikal]

Hi Julia, 

I'm using the Brunello library (Human CRISPR knockout library),  I did the FACS sorting today and collected the top 4% cells ( 2 million cells ), I started with 50 million which is equivalent to  700x representation of the library. I  was wondering if I should grow them to 50 million ( to maintain the library representation)  then harvest the gDNA as this what is written in step 57 in the  protocol      or I'm understanding that wrong and i should harvest the gDNA from these 2 million cells as your reply in the previous email is 2% should be sufficient? 

Thank you so much!
Rehab  

[Julia Joung]

Hi Rehab,

Yes, you can just harvest the gDNA from the 2 million cells without growing them up to 50 million. Next time you do the FACS screen though, make sure to collect the bottom 4% of cells as well for the control arm. It will give you better signal than normalizing the top 4% to the unsorted cells.

Best,

Julia

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