title: Sample 1
organism: Canis lupus familiaris
description: Protocols: Healthy canine lung tissues were collected from either privately-owned dogs euthanized for reasons unrelated to this study, or from a healthy lung region of lobes resected for a solitary lung tumor. Samples were collected with informed owner consent and under the local Animal Ethics Committee approval (#20-2245). In each dog, one lung biopsy was collected in the alveolar region directly after death or lobectomy and transported in HBSS (Gibco) containing 5% v/v of FBS (Gibco) on ice for immediate processing. Retrospectively, only histopathologically confirmed cases free of lung disease at the biopsy site were retained for the present analysis. Each lung sample underwent mechanical dissociation with razor blades and was suspended in HBSS + 5% FBS with collagenase A (1 mg/mL; Sigma) and DNase I (0.05 mg/mL; Roche) before incubation at 37°C for 45 min. The cells were then filtered through a 70 µm cell strainer (BD Falcon) and resuspended in PBS (Biowest) containing 10 mM EDTA (Merck Millipore). Red blood cells were lysed as needed with a lysis buffer containing 0.15 M NH4Cl, 0.01 M KHCO3 and 0.1 mM EDTA at pH 7.5. The final cell suspension contained between 500 and 1000 cells/µL in PBS containing 0.04% of BSA (Sigma) and 0.2 U/µL of RNase inhibitor (Roche). Final cell viability was assessed by trypan blue staining and considered acceptable above 70%. Approximatively 10,000 cells from each lung sample were loaded in a Chromium Controller or Chromium iX instrument (10x Genomics, Pleasanton, CA) and encapsuled with unique barcoded primers using the drop-sequencing method according to manufacturer's instructions.  Emulsion breakage, cDNA amplification and libraries construction were performed using Chromium Single Cell 3′ reagent kit v2 (10x Genomics) according to manufacturer's instructions.