GSM8240031: Lung, yak rep1, scRNAseq; Bos grunniens; RNA-Seq
Following aseptic procedures, for each individual, lung tissue samples of 1 cm³ in size were precisely collected using surgical scissors from 6 sampling points on both sides of the lung lobes (i.e. left anterior, left middle, left posterior, right anterior, right middle and right posterior lobes), located 5 cm from the lung apex, avoiding bronchial tubes and blood vessels as much as possible. Immediately, each piece of lung tissue was transferred separately to a freezing tube containing cell freezing medium (90% FBS and 10% DMSO) and thoroughly resuspended. For each animal, lung tissue samples collected from 6 different sites were pooled for subsequent single-cell transcriptomic analysis. Lung tissues in cryovials were thawed in a 37 °C water bath and washed 3 times in RPMI 1640 medium. The lung tissues were finely minced into approximately 0.5 mm³ fragments by using sterile surgical scissors in RPMI 1640 medium. These fragments were digested at 37 °C for 30 min in 1 mg/mL Type I collagenase, followed by 10 min of digestion at 37 °C in 0.25% trypsin-EDTA. Lastly, 10% foetal bovine serum was added to stop digestion. Lung tissue suspensions were filtered through a 40 μm cell strainer, and erythrocytes were removed with red blood cell lysis buffer. Lung cells were resuspended in DPBS containing 2% foetal bovine serum, and the cell suspension was counted and stored for subsequent scRNA-seq. Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10× Genomics). In brief, the whole library construction process was divided into 3 steps: GEM Generation & Barcoding, Post GEM-RT Cleanup & cDNA Amplification and 3' Gene Expression Library Construction.