Transcriptomic and topological analyses of human lung tumors identify cancer-associated fibroblast subsets associated with T cell exclusion

We report on the application of focused stromal cell single cell sequencing on paired adjacent and NSCLC tissue from 15 patients. Overall design: Stromal cells were sorted from single cell suspensions of NSCLC and adjacent lung tissue samples then sequenced using the 10X genomics platform. 15 tumor and 12 adjacent tissue samples were analyzed. The adjacent lung and tumor tissue were weighed and cut into sections of 0.1-0.2 grams then placed into 5 mL microtube (Argos Technologies). Sections were minced with scissors and enzymatically digested in CO2-independent media (Fisher Scientific, 18045088) with Collagenase IV 0.25mg/ml (Sigma-Aldrich, C5138-1G), Collagenase D 200 U/ml (Sigma-Aldrich, 11088882001), and DNAse 0.1 mg/ml (Sigma-Aldrich, DN25-1G) for 40 minutes at 37°C under 80 rpm agitation. Cell suspensions were passed through a syringe with an 18-gauge needle 8-10 times, filtered through a 70µm cell strainer, then lysed in red blood cell (RBC) buffer (Fisher Scientific, NC9067514). The cells were resuspended in buffer comprising of DPBS (Corning, D8537-6X500ML) with 5% BSA (Equitech-Bio, BAH62-0500) and 1mM EDTA (Sigma-Aldrich, 46-034-CI) then counted using hemocytometer and Trypan blue (Fisher Scientific, MT25900CI). Raw FASTQ files and processed feature-barcode matrices, containing UMI counts in Market Exchange Format.