GSM8669809: d80 CF, distal lung, sample 3; Ovis aries; RNA-Seq
i. For single cell isolation from proximal or distal lung ~300 mg of tissue was cut into 2-3 mm pieces and subjected to two rounds of digestion.  First in 5ml of collagenase solution (200 U/ml collagenase, 40 IU/ml DNase I  (all from Worthington), 3 mM CaCl2, at 37°C, with continuous stirring for 30-40 min. Alternatively, Roche Liberase TL (Millipore Sigma) containing 100 µg/ml collagenase was used.  Tissue clumps were then disassociated by pipetting 10 times in a 5 ml serological pipette and left to settle for 3-4 min at which point the supernatant was collected and stored on ice as cell suspension 1. Second, 5 ml of fresh collagenase digestion solution as above was added to the settled tissue clumps remaining after the first digestion and again stirred at 37°C for 30-40 min, at which point very little tissue debris remained. The supernatant (cell suspension 2) was pooled with cell suspension 1 and together passed through a cell strainer (200 µm, pluriSelect) to remove any tissue debris. The strained cell suspension was then centrifuged at 300 x g for 5 min, washed once in HBSS (Sigma Aldrich) + 2% FBS and centrifuged again. 4 ml of 1× Red Blood Cell Lysis Solution (Miltenyi Biotec 130-094-183) was added to the cell pellet which was vortexed for 5 seconds and then incubated for 5 minutes at room temperature. After 10 ml of HBSS with 2% FBS was added, the mixture was centrifuged at 300 × g for 5 min and the supernatant was removed. The cell pellet was then digested with 3 ml of Accutase (Stem Cell Technologies, #AT104) for 15-25 min with frequent gentle pipetting with a wide-bore pipette after which time cells were visualized under a microscope. If any cell clumps persisted these were collected by centrifugation and further digested with 3 ml of Accutase for an additional 10-15 min. Accutase was removed by washing the cells with 5 ml HBSS + 2% FBS, followed by centrifugation to collect the cells. The supernatant was discarded, and the cell pellet was resuspended in 0.5-1.0 ml HBSS, passed through a 20-µm mini cell strainer (pluriSelect) and cells counted. ii) Single-cell RNA-sequencing and analysis ii) Approximately 3000- 5000 cells per tissue sample were used for scRNA-seq using the 10x Genomics Chromium Single Cell 3' Reagent Kit v3, or v3.1.  After quality control of both cDNA and final libraries using Tapestation, the libraries were sequenced on a NovaSeq 6000 machine. Reads were aligned to the Oar_v4.0/oviAri4 (Texel) genome using Cell Ranger 3.1.0. Cells were filtered for quality using cuts for standard metrics: library size, number of detected genes and mitochondrial read percentage. Ribosomal protein genes were also filtered out. The resulting objects were normalized and batch-corrected using the Seurat R package, version 5.1.0, followed by clustering and UMAP (Uniform Manifold Approximation and Projection) dimensionality reduction. Seurat was also used to find cluster markers by performing differential gene expression analysis between clusters using the receiver operating characteristic (ROC) method. Libraries generated from single cells according to the manufacturer's protocol for 10X Genomics Chromium Single Cell 3' Reagent Kits v3 or 3.1.