Single-cell RNA sequencing of airway submucosal glands in wild-type and cystic fibrosis Pigs

Submucosal glands (SMGs) in the upper respiratory system of mammals play important roles in fluid secretion, mucociliary transport, and host defense. In people with cystic fibrosis (CF), airway mucus obstruction and SMG duct occlusion are hallmark features. Although intensive investigations have focused on airway surface epithelium in normal and diseased lungs, our knowledge of SMGs is limited in terms of cell biology and physiology. Here, we conducted single cell RNA sequencing (scRNA-seq) of airway SMGs in newborn piglets. First, we dissected individual SMGs and surrounding non-epithelial tissues from newborn non-CF (n = 4) and CF (n = 4) pig trachea. Second, we did scRNA-seq using 10x genomics chromium platform. Third, we processed the raw sequencing data using an improved RefSeq pig genome reference. Last, we integrated all samples together and clustered cells into 14 major cell clusters using Seurat R toolkit (Stuart et al., 2019). These data allowed us to elucidate airway SMGs at single cell resolution. Moreover, we found that cell-types and gene expression were the same in non-CF and CF SMGs, suggesting that loss of epithelial anion secretion rather than an intrinsic cell defect causes CF mucus abnormalities. We dissected and collected individual tracheal SMGs from 4 wild-type (WT) and 4 CFTR-/- (CF) newborn pigs.To eliminate contamination of airway surface epithelial cells, we brushed off airway surface epithelial cells as much as possible. To explore the SMGs before dissection, we peeled the epithelial layer away from the cartilage layer of the trachea. Single cells were processed using the 10X Genomics Chromium controller. cDNA libraries were generated using the Chromium Single cell 3’ V2 kit. Sequencing was conducted on a HiSeq 4000 system from Illumina.