Mouse tumor dissociation buffer

For scRNAseq, randomly selected lymph node samples (1 sample from CpG control group and 2 samples from TOP2A vaccine treated group) and the breast tumor samples (1 sample from CpG control group and 1 sample from TOP2A vaccine treated group) of C3(1)/Tag mice were harvested at the end of the study, minced and digested at 37 °C for 30 minutes with mouse tumor dissociation buffer (Miltenyi Biotec, CA) to generate single-cell suspensions per the manufacturer’s instructions. The processed samples were directly stained with violet viability dye, APC anti-CD45, and CD45+ leukocytes were sorted using FACS. FACS-sorted CD45+ leukocytes were then spun down at 300 g for 5 minutes and counted manually with a Neubauer Chamber. Approximately 2.0 × 10⁴ cells were loaded onto the 10X Chromium Controller per the manufacturer’s instruction, resulting in a recovery of about 1 × 10⁴ cells. For the lymph node samples, the libraries of single-cell transcriptome were generated by Chromium Single Cell 3’ v3 Reagent Kits (10x Genomics). For tumor samples, single-cell transcriptome and single-cell TCR libraries were prepared using a 10x Chromium Single-cell 5′ and VDJ library construction kit. All of the libraries were sequenced using NextSeq 500/550 High Output Kits v2 (150 cycles) (Illumina) according to the manufacturer’s protocol.

For scRNA-seq, B[a]P-induced lung tumors from the second experiment were harvested and pooled from different treatment groups at the end of the study, then minced and digested at 37 °C for 20 min with mouse tumor dissociation buffer (Miltenyi Biotec, Auburn, CA, USA) to generate single-cell suspensions per the manufacturer’s instructions. The lung tumors were separated from the adjacent normal tissue before being pooled, and about five tumors were pooled from each mouse for scRNA-seq. CD45 is a transmembrane protein tyrosine phosphatase located on most nucleated hematopoietic cells; CD45 is used as the marker to differentiate immune cells from other non-immune epithelial and stromal cells. Single-cell suspensions were stained with CD45 surface markers, and the CD45⁻ single cells were flow-sorted and then spun down at 300× g for 5 min and counted manually with a Neubauer chamber. Approximately 1.6 × 10 [4 (link)] cells were loaded onto the 10× Chromium Controller per the manufacturer’s instructions. ScRNA-seq libraries were generated by Chromium Single Cell 3′ v3 Reagent Kits (10× Genomics, Pleasanton, CA, USA) and sequenced using NextSeq 500/550 High Output sequencing reagent Kits v2 (150 cycles) (Illumina) according to the manufacturer’s protocol. There were two replicates for each of the experimental groups (control, Mito-HNK, Mito-LND, combination).

For scRNA‐seq, right middle lung lobes were harvested from mice in each treatment group at the end of the study, then minced and digested at 37 °C for 30 min with mouse tumor dissociation buffer (Miltenyi Biotec, CA) to generate single‐cell suspensions per the manufacturer's instructions. TILs were directly stained with violet viability dye, APC anti‐CD45, and CD45⁺ leukocytes were FACS sorted out. FACS‐sorted CD45⁺ leukocytes were then spun down at 300 × g for 5 min, counted manually with Neubauer Chamber. Approximately 2.0 × 10⁴ cells were loaded onto the 10x Chromium Controller per the manufacturer's instruction, resulting in a recovery of about 1 × 10⁴ cells. The scRNA‐seq libraries were generated by Chromium Single Cell 3' v3 Reagent Kits (10x Genomics) and sequenced using NextSeq 500/550 High Output Kits v2 (150 cycles) (Illumina) according to the manufacturer's protocol. There were two replicates for each experimental group.