GSM9067059: Combo-Replicate-3; Mus musculus; RNA-Seq
At the time points, tumors were harvest under sterile conditions and weighted. Single-cell suspensions were obtained by enzymatic digestion, following the MACS tumor dissociation kit instructions (cat. #130-096-730; Miltenyi Biotec) and the number of viable cells counted Single-cell RNA sequencing was conducted using the 10X Genomics Chromium System (10X Genomics, Pleasanton, CA) by the Molecular Genomics Core at Moffitt Cancer Center and Research Institute. Following tissue harvest and dissociation, single-cell suspensions were prepared, washed twice with 1X PBS (calcium- and magnesium-free) containing 0.04% BSA (w/v), and resuspended in the same buffer according to the 10X Genomics cell preparation protocol. Cell viability and concentration were assessed using acridine orange/propidium iodide (AO/PI) dual fluorescent staining on the Nexcelom Cellometer K2 (Nexcelom Bioscience LLC, Lawrence, MA). Cells were then loaded onto the 10X Chromium Single Cell Controller at a concentration of 1,000 cells/µL to target recovery of approximately 10,000 cells per sample. Briefly, individual cells, reagents, and gel beads were encapsulated into nanoliter-sized Gel Beads-in-Emulsion (GEMs), and reverse transcription of polyadenylated mRNA occurred within each droplet at 48°C. Gene expression (GEX) libraries, T-cell receptor (TCR) V(D)J enrichment, and VDJ libraries were generated according to the Chromium GEM-X Single Cell 5' v3 Gene Expression User Guide. Sequencing was performed on the Illumina NovaSeq 6000 platform, generating approximately 50,000 reads per cell. Data processing—including demultiplexing, barcode assignment, alignment, and gene counting—was carried out using Cell Ranger v9.0.0 (10X Genomics). Visualization and downstream analysis were performed with the 10X Genomics Loupe Browser v8.1.2.