CD8+ T-cells Cells were electroporated at 20e6 cells per cuvette using the pulse code EH115 . The total number of cells for electroporation
was scaled as required. Immediately after electroporation, 1mL of pre-warmed media was added to each cuvette and cuvettes were
placed at 37 degrees for 20 minutes. Cells were then transferred to culture vessels in X-Vivo media containing 50U/mL IL-2 at
1e6 cells /mL in appropriate tissue culture vessels. Cells were expanded every two days, adding fresh media with IL-2 at 50U/mL
and maintaining the cell density at 1e6 cells /mL.
Assembled ribonucleotide proteins (RNPs) were dispensed into a 96W V-bottom plate at 3mL per well. Cells
were spun down, resuspended in Lonza P3 buffer at 1e6 cells per 20mL, and added to a V-bottom plate with RNPs. The cells/RNP
mixture was transferred to a 96 well electroporation cuvette plate (Lonza, cat #VVPA-1002) for nucleofection using the pulse code
EH115. Immediately after electroporation, 80mL of pre-warmed media was added to each well and incubated at 37C for 20 minutes.
48 hours after transduction, 1e6 cells were resuspended in P3 buffer and 3 mL of Cas9 (Stock 40 mM) was added. Cells were transferred
to a 96 well electroporation cuvette plate (Lonza, cat #VVPA-1002) for nucleofection using the pulse code EH115. 24 hours post
nucleofection, cells were treated with 2.5ug/mL Puromycin for three days, and subsequently sorted for live cells using Ghost Dye 710
(Tonbo Biosciences, Cat #13-0871).