Spinfection protocol

[Nina Stahel]

Hello,

I have a question regarding the spinfection protocol used in "Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells" (Science 343, p84-87, 2014).

After seeding the cells into the 12-well plate (3x10⁶/well), do you first leave them overnight to settle and attach, or do you immediately add the virus and spin down the cells and the virus together? I'm asking because upon seeding this amount of cells into a 12-well plate, the cells tend to overlay, which might not equally expose them to the virus if added on the already attached cells.

Thank you and regards

[Neville Sanjana]

Hi Nina,

For the spinfection, we immediately spin the cells after placing them (and virus) into the 12-well. We normally spinfect in the afternoon, place them in the incubator overnight and then split out the next morning into appropriately sized flasks. During the spinfection, the cells are equally exposed to the virus because the plates are being spun, which resuspends the cells. 

Hope that helps,

Neville

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[Nina Stahel]

Hi Neville,

That's very helpful, thank you very much!

Regards
Nina

[Nina Stahel]

Follow-up question:

If you have to apply puro selection, do you also split the cells out into flasks the day after spinfection and perform the puro selection in the flasks?

Thanks again and kind regards from Switzerland

[Neville Sanjana]

Hi Nina,

That's correct. We normally spinfect in 12-well dishes (day 1 afternoon), leave in the incubator overnight in 12-well dishes, split out into flasks (day 2 morning), and then add puromycin in flasks (day 2 afternoon, 24 hours after spinfection). This workflow originates from the Broad's RNAi/GPP Platform and we have found it works well for a wide variety of screens.

Best wishes,

Neville

[jian...@gmail.com]

Hi Neville,

One quick question, in the Science paper "Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells" (Science 343, p84-87, 2014), it says "The 12-well plate was centrifuged at 2,000 rpm for 2 h at 37°C. After the spin, media was aspirated and fresh media (without polybrene) was added."

I will assume that after spin, the media was replaced with the regular complete media without polybrene and "without virus", right?


Thanks
Jianke

[Neville Sanjana]

Hi Jianke,

That's correct. Fresh media (without polybrene or virus) is used after the spinfection. The idea is that infection happens during spinfection so that those elements are no longer necessary.

Best,

Neville

[Deathcrafter 2000]

Can you just leave the cells with the virus (and polybrene) overnight after spinfection, and not remove the media to minimise loss of cells?