Sentinel p16INK4a+ cells in the basement membrane form a reparative niche in the lung [scRNA-Seq]

A single-cell transcriptional analysis was performed on p16INK4a+ cells from the adult murine lung. Whole adult murine lung tissue was dissociated to single cells and subjected to fluorescence activated cell sorting (FACS) to select all live p16INK4a + cells. The single cell RNA-sequencing library was then subsequently generated. Cells were sequenced at a depth of ~30,506 mean reads/cell. We captured approximately 8752 cells with a mean of 2457 genes detected per cell utilizing a droplet-based barcoding approach to capture single cells for RNA sequencing. The results demonstrate that the p16INK4a + population are predominantly in the immune CD45+ populations and PDGFRa+ mesenchyme. Overall design: The INKBRITE animals we constructed by inserting a tandem cassette of H2B-GFP expressed in frame with the p16INK4A gene product in the murine Cdkn2a locus into a bacterial artificial chromosome (BAC). The expression of multiple copies of a stable fluorescent protein is driven by p16INK4A promoter. Whole adult murine lung tissue was dissociated to single cells stained with DAPI for live cells selection and subjected to FACS to select live GFP+ (p16INK4a+) cells. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Live Gp16INK4a+ cells were prepared using the 10X single cell instrument and v1 10X single cell kits according to standard protocol. Single cell sequencing of adult murine lung GLI2+ population was performed by using the HiSeq2500.