scRNA-seq Analysis of BaP-Induced Lung Tumors

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).