Nextseq 500 550 high output sequencing reagent kits v2

B[a]P and tricaprylin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Chromium Single Cell 3′ v3 Reagent Kits (10x Genomics) and NextSeq 500/550 High Output sequencing reagent Kits v2 (150 cycles) (Illumina, San Diego, CA, USA) were used according to the manufacturer’s protocol [9 (link),10 (link),11 (link)]. LKR13 cells, a mouse lung adenocarcinoma line that expresses mutant KrasG12D on the SV129 background, were a generous gift from Dr. Jonathan M. Kurie (MD Anderson Cancer Center, Houston, TX, USA). LKR13-Luc cells were generated by transfecting LKR13 cells with a lentivirus expressing firefly luciferase (PLV-10064-200; Cellomics Technology, LLC, Halethrope, MD, USA). H2030-BrM3 cells were a generous gift from Dr. Joan Massage (Memorial Sloan Kettering Cancer Center, New York, NY, USA). All cell lines were grown in RPMI (Roswell Park Memorial Institute) 1640 medium (Cat# 11875; Thermo Fisher Scientific, Wiltham, MA, USA) supplemented with 10% fetal bovine serum at 37 °C in 5% carbon dioxide. A/J mice, FVB mice, and SV129 mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Mito-HNK and Mito-LND were synthesized as previously described [4 (link),8 (link)] (Figure 1A).

Benzo[a]pyrene (B[a]P) and tricaprylin were purchased from Sigma Chemical Co. (St. Louis, MO). NLE was purchased from BIOO Scientific (Austin, TX). The let‐7b mimic was purchased from Dharmacon. Using proprietary technology, the mimic was chemically enhanced to preferentially program RNA‐induced silencing complex (RISC) with an active miRNA sequence (https://horizondiscovery.com/‐/media/Files/Horizon/resources/Product‐inserts/miridian‐mimics‐inhibit‐neg‐contr‐prodinsert.pdf). Fluorescent let‐7b‐cy5 used in ex vivo imaging studies was obtained from Dharmacon. The miRNeasy mini kit, miScript II RT Kit, miScriptSyBr Green PCR Kit, hsa‐let‐7b primer, and small nuclear RNA U6 were purchased from Qiagen (Valencia, CA). Chromium Single Cell 3′ v3 Reagent Kits (10x Genomics) and NextSeq 500/550 High Output sequencing reagent Kits v2 (150 cycles) (Illumina) were used 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).