GSM8487172: STING agonist, Cd45 positive cells, scRNAseq; Mus musculus; RNA-Seq
KPC tumors were surgically resected. Tumors were excised and mechanically dissociated into smaller fragments. These fragments underwent enzymatic digestion with LiberaseTM (300 μg/mL) and mechanical dissociation using the GentleMACS Octo dissociator (protocol: hard tumor dissociation, TDK-3, 1 hour at 37°C). Following dissociation, the cell suspension was filtered using a 70 μm strainer and then washed twice with RPMI medium at 400 rcf for 5 minutes. After dead/live cell staining and blocking non-specific binding using anti-FcRII/III (Biolegends) for 10 minutes, cells were stained with anti-CD45 antibody for 1 hour at 4C. Live CD45+ leukocytes were sorted by CytoFLEX SRT (Beckman). Library generation was performed according to the manufacturer's instruction (Chromium GEM-X Single Cell 5' Reagent Kits v3, 10X Genomics). Briefly, Cd45 positive cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM).   Poly-adenylated mRNA contained in each single GEM was reverse transcripted to cDNA that includes an Ilumina R1 primer, Unique Molecular Identifier (UMI), and 10X barcode.  The barcoded cDNA was cleaned up with Dynabeads MyOne SILANE, amplified by PCR, and appropriate size fragments were selected with SPRI select.  For gene expression library construction, Ilumina R2 primer, paired-end constructs with P5 and P7, and sample index were added.