Single-nucleus RNA sequencing of ex vivo precision-cut lung slices from normal human lung tissue

This study aimed to elucidate how epithelial–macrophage interactions contribute to pulmonary fibrosis. Using bleomycin-induced and genetic mouse models (Mif-2 KO, SPC-Cul3 KO, SPC-Mif KO, CX3CR1-Cd74 KO, PF4-Cd74 KO, and TGF-ß transgenic mice), we performed single-cell transcriptomic profiling of lung cells and compared them with wild-type controls. Overall design: Samples were inflated by injecting 3% low-melting agarose followed by cutting samples into 5x5 mm cubes. Peripheral slices (300 µm) were cut with a vibratome. Sections were placed into 1 mL 10% DMEM with 1% pen-strep in a 24 well plate. hPCLS were washed with HBSS x 2 h, rocking, at 37 degrees, and then treated with vehicle (DMSO) or Fibrotic cocktail (FC) [5 ng/ml recombinant transforming growth factor-ß (TGF-ß) (240-B, R&D Systems), 5 µM platelet-derived growth factor-AB (PDGF-AB) (PHG0134, GIBCO), 10 ng/ml tumor necrosis factor-a (TNF-a) (210-TA, R&D Systems), and 5 µM 1-Oleoyl lysophosphatidic acid (LPA) (62215, Cayman Chemical)] +/- 50µM 4-IPP drugs in 500 ul of 0.25% DMEM w 1% pen-strep. Every 2 days, the media was changed and treated again. At the end of the 5 days, PCLS were washed in cold PBS x 2 then placed in RNAlater solution (Thermo). Nuclei were isolated from frozen tissue and profiled with snRNAseq.