CD8-positive, alpha-beta memory T cell, Homo sapiens modified with CRISPRko Sp-Cas9, transduced (lentivirus) with a guide library (MOI of 0.4)
CD8+ T cells were activated with CD3/CD28 dynabeads at a 1:1 ratio. At 24 hours post-activation, CD8+ T cells were transduced with gRNA lentivirus encoding a GATA3 targeting gRNA. At 48 hours post-activation, 3.5 x 10^5 T cells were electroporated with Cas9 protein. Briefly, cells were collected, spun down at 90xg for 10 minutes, resuspended in 20uL of Lonza P3 Primary buffer with 1.12 ug Cas9, and electroporated with the pulse code EH115. Cells were cultured in PRIME-XV T cell expansion XSFM (FujiFilm) supplemented with 5% human platelet lysate (Compass Biomed), 100 units/mL of penicilllin, 100 units/mL of streptomycin, and 100 units/mL human IL-2 (Peprotech).
T cells were maintained at a concentration of 1-2e6 per mL for 9 days before RNA-sequencing.
RNA was harvested using Norgen's Total RNA Purification Kit.
RNA libraries for RNA-seq were prepared by Azenta Life Sciences.
charles-gersbach:cd8-tcells-primary-cell-cas9-ko-gata3-rep2