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.Th1样细胞按照前文所述方法制备，起始实验条件为：每例供体取8×10⁵份新鲜分离的初始CD4⁺ T细胞，重悬于10 mL基础培养基中，该培养基添加Th1极化混合液与CD3/CD28磁珠。经分化培养后，供体1、2、3、4的细胞收获量分别为23.4×10⁶、21.75×10⁶、14.46×10⁶及19.05×10⁶。随后将各组细胞分别浓缩至1 mL体积，所用缓冲液为含2%胎牛血清（FBS）、1%青链霉素混合液（P/S）、2 mM乙二胺四乙酸（EDTA）的磷酸盐缓冲液（PBS）；每样本加入10 μL生物素标记抗CD30抗体（BD Biosciences，货号：555828）。与该生物素化抗体孵育20分钟后，将细胞洗涤两次，随后严格遵循制造商的实验方案，分离得到磁珠游离的CD30富集组分与CD30耗竭组分细胞（STEMCELL Technologies，货号：17653）。此次分选后，供体1的耗竭组分细胞数为13.2×10⁶，富集组分细胞数为3.72×10⁶；供体2的耗竭组分与富集组分细胞数分别为10.5×10⁶与2.9×10⁶；供体3的耗竭组分与富集组分细胞数分别为8.43×10⁶与2.81×10⁶；供体4的耗竭组分与富集组分细胞数分别为11.1×10⁶与3.63×10⁶。从上述各组分选取1×10⁶个细胞，分别接种于1 mL基础培养基中，一组添加CD3/CD28磁珠（磁珠与细胞比例为1:8），另一组不添加磁珠，孵育24小时。孵育24小时后，使用DynaMag-2磁分离架（Invitrogen，货号：12321D）移除磁珠，对细胞进行计数；随后从每例供体的对应组中取2×10⁵个细胞，按分组混合为四组：CD30⁻未刺激组、CD30⁻刺激组、CD30⁺未刺激组、CD30⁺刺激组。将各组细胞通过40 μm细胞筛过滤后计数，随后从每组中取5.5×10⁴个细胞，上样至Chromium Next GEM Chip K芯片的4个独立通道中。本次实验的基因表达文库构建严格遵循制造商说明书，使用Chromium Next GEM单细胞5'端试剂试剂盒v2（双索引版）完成。随后将文库置于NovaSeq S2测序流动槽进行测序。