title: GSM9151221: scRNA-seq,M-TNF,ADT; Mus musculus; OTHER
library_construction_protocol: Splenic CD8⁺ T cells were enriched using the EasySep™ Mouse CD8⁺ T Cell Isolation Kit (STEMCELL Technologies, cat. #19853) according to the manufacturer's instructions. Enriched cells were stained with Live/Dead Fixable Aqua Dead Cell Stain (Invitrogen, cat. #L34966; 1:1000 dilution in PBS) for 5 minutes at room temperature. A surface antibody cocktail (1:300 final dilution) was prepared in 1% BSA/PBS and supplemented with TruStain FcX™ PLUS (anti-mouse CD16/32, BioLegend, cat. #156604; 5 μL per sample). 25 μL of this cocktail were added to each sample, followed by a 10-minute incubation at 4°C. Subsequently, 25 μL of a TotalSeq™-C antibody cocktail (1:50 final dilution), along with a TotalSeq™-C Hashtag antibody (0.2 μg per sample), were added and samples were incubated for 30 minutes at 4°C. Details of the antibodies used are provided in Table 1. To minimize aggregate formation, all antibody cocktails were centrifuged at 14,000 × g for 10 minutes at 4°C prior to use. After staining, cells were washed once in 1% BSA/PBS and live CD3e⁺CD8a⁺ T cells were sorted using the Aurora Cell Sorter. Post-sort, cells were counted and assessed for viability. The final suspension was adjusted to 1,300 cells per μL, and equal numbers of cells from up to four samples were pooled prior to library preparation 45k cell per pooled sample were subjected to droplet-based massively parallel single-cell RNA sequencing. cDNA was prepared after the GEM generation and barcoding, followed by the GEM-RT reaction and bead cleanup steps. Purified cDNA was amplified for 11-16 cycles before being cleaned up using SPRIselect beads.  Samples were then run on a Bioanalyzer to determine the cDNA concentration.  V(D)J target enrichment (TCR) was performed on the full-length cDNA.  Gene Expression, Enriched TCR and Feature libraries were prepared as recommended by the 10x Genomics Chromium GEM-X Single Cell 5' Reagent Kits User Guide (v3 Chemistry Dual Index) with Feature Barcoding technology for Cell Surface Protein and Immune Receptor Mapping user guide, with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform, the Chromium GEM-X Single Cell 5' Kit v3, 16 rxns (PN-1000699), Chromium GEM-X Single Cell 5' Chip Kit (PN-1000698), Chromium Single Cell Mouse TCR Amplification Kits (PN-1000254), Dual Index Kit TT Set A, 96 rxns (PN-1000215), Chromium GEM-X Single Cell 5' Feature Barcode Kit v3, 16 rxns (PN-1000703) and Dual Index Kit TN Set A, 96 rxns (PN-1000250) were used.  The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer's protocol (KAPA Biosystems/Roche) to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument.  Normalized libraries were sequenced on a NovaSeqX plus S4 Flow Cell using the XP workflow and a 151x10x10x151 sequencing recipe according to the manufacturer protocol.  A median sequencing depth of 50,000 reads/cell was targeted for each Gene Expression library and 5000 reads/cell for each V(D)J and Feature library.