RNP nucleofection for cell lines using Lonza 4D Nucleofector
Bring 100 pmol of Cas9 to a final volume of 5 µL using Cas9 buffer (20 mM HEPES-KOH pH 7.5, 150 mM KCl, 10% glycerol, 1 mM TCEP).
For 40 µM stock: 2.5 µL.
Bring 120 pmol sgRNA to a final volume of 5 µL using Cas9 buffer.
This means you will need a minimum sgRNA concentration of 24uM.
Add Cas9 to sgRNA slowly while swirling pipette tip, should take 30s to 1 minute.
Allow RNP to form for 10-20 minutes.
Count cells. (Trypsinize as needed.)
For each nucleofection, pipette 200k cells into a 15 mL conical.
Spin 100 x g for 10 minutes to pellet cells softly.
While the cells are spinning, prepare plate and cuvette.
Prepare a 12-well-plate with 1mL media per well, and pre-warm in the incubator.
Prepare and label wells on 20uL nucleofection strips.
Configure Lonza 4d using recommended cell-type program.
Pipette off media from cells, gently but completely, using a P200.
The pellet is very soft so be careful.
Resuspend cells in 20 µL of nucleofector solution (usually SF media) using a P200.
Add the entire 10 µL RNP mix to the 20 µL resuspension and mix.
Add 1uL of 100uM donor DNA (100 pmoles) and mix well.
Add nucleofection mixes to the multiwell cuvette, and cap.
Pay attention to the orientation of the cap and cuvette in the nucleofector, which is noted in the manufacturer’s instructions.
Insert cuvette into nucleofector and zap.
Allow cells to sit in nucleofection strips for 10 minutes post-nucleofection.
This is supposed to increase efficiency.
Add 80uL of pre-warmed media to each well.
Pipette mixture out with a P200 into your pre-warmed 12-well plate.
This should get the vast majority of cells, but if you wish, you may wash out the rest with media from the same well, chemistry-style.
Allow cells 24 hours to settle and recover before attempted downstream analysis.
Consider including un-zapped controls to test viability.
