| Experiment Id | GSE102591 | Name | Hedgehog gradient in the stroma maintains niche diversity and organ function [Drop-seq] |
| Experiment Type | RNA-Seq | Study Type | Baseline |
| Source | GEO | Curation Date | 2023-07-21 |
| description | A single-cell transcriptional analysis was performed on GLI2+ stromal 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 GLI2+ cells. The single cell RNA-sequencing library was then subsequently generated. Cells were sequenced at a depth of ~60,000 reads/cell. We captured approximately 4,600 cells with a median of 2,246 genes detected per cell utilizing a droplet-based barcoding approach to capture single cells for RNA sequencing. The results demonstrate that the GLI2+ population is segregated into distinct stromal subtypes along the proximal-distal axis of the lung with distinct functional contribution to matrix production and paracrine signaling within their distinctive niche. The CreERT2-2A-tdTomato expression cassette was inserted into the Gli2 locus to generate a murine tool that allows us to isolate GLI2+ cells by endogenous tdTomato reporter expression. Whole adult murine lung tissue was dissociated to single cells, stained with anti-CD45-PE-Cy7, anti-Epcam-BV421 and DAP, and subjected to FACS to select live GLI2+ cells. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. The hematopoietic cells were excluded by stained with anti-CD45-PE-Cy7 and the epithelial cells were excluded by stained with Epcam-BV421. Then the GLI2+ cells were sorted by endogenous tdTomato fluorescence. The live GLI2+ 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. |
