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

We collected freshly sorted mesenchyme (live CD45-Epcam-CD31-PDGFRa+GFP- or GFP+) from dissociated lung tissue of INKBRITE animals to characterize the transcriptome difference between p16INK4a+ vs p16INK4a- cells at homeostasis and during injury. We used fluorescence activated cell sorting (FACS) to exclude immune (CD45+), epithelial (Epcam+), and endothelial (CD31+) cells and positively select for PDGFRa+ mesenchyme. Cells were sequenced at a depth of average of 45 million reads/sample. The results revealed an increase of senescence associated secretory phenotype (SASP) signature in p16INK4+ cells that becomes refined after injury. We found the expression of an epithelial growth factor epiregulin (ereg) increased in p16INK4a+ cells after injury suggesting a possible role of p16INK4a+ senescence during regeneration of lung epithelium. 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 CD45-Epcam-CD31-PDGFRa+GFP+ (p16INK4a+) and CD45-Epcam-CD31-PDGFRa+GFP- (p16INK4a-) cells. We collected p16INK4a+ and – cells from 3 INKBRITE naphthalene injured animals and 3 solvent controls. The RNA was extracted and shipped to GENEWIZ company for sequencing o the Illumina HiSeq instrument. Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software