Second scRNA-sequencing run (Th1-like, CD30) - Raw (MTX) & Processed (RDS) data

Th1-like cells were generated as described in the previous sections (starting condition: for each donor, 8x105 freshly isolated naive CD4+ T cells were resuspended in 10 mL base media supplemented with Th1 cocktail and CD3/CD28 beads). The differentiation period resulted in 23.4x106 cells for donor 1, 21.75x106 cells for donor 2, 14.46x106 for donor 3, and 19.05x106 cells for donor 4. The cells were then concentrated into 1 mL volume each in 2% FBS 1% P/S 2 mM EDTA PBS, and 10 µL anti-CD30-Biotin was added to each sample (BD Biosciences, P/N 555828). After 20 minutes of incubation with the biotinylated antibody, the cells were washed twice and then the manufacturer’s protocol was followed to obtain magnetic particle free CD30 enriched and CD30 depleted fractions of cells (STEMCELL Technologies, P/N 17653). This process resulted in 13.2x106 depleted cells and 3.72x106 enriched cells for donor 1, 10.5x106 depleted and 2.9x106 enriched cells for donor 2, 8.43x106 depleted and 2.81x106 enriched cells for donor 3, 11.1x106 depleted and 3.63x106 enriched cells for donor 4. 1x106 cells from each of these fractions were plated in 1 mL base media with and without CD3/CD28 beads at a ratio of 1 bead per 8 cells for 24 hours. After 24 hours, the beads were removed using a DynaMag-2 Magnet (Invitrogen, P/N 12321D), cells were counted, and 2x105 cells from each donor were combined into pools for each condition: CD30-, CD30- stimulated, CD30+, CD30+ stimulated. Cells from each condition were strained through 40 µm cell strainers, counted, and then 5.5x104 from each condition loaded into 4 individual lanes of a Chromium Next GEM Chip K. The gene expression library for this run was constructed as per the manufacturer’s protocol using Chromium Next GEM single Cell 5’ Reagent Kits v2 (Dual Index). The run was sequenced on a NovaSeq S2 flow cell.