Hedgehog gradient in the stroma maintains niche diversity and organ function [RNA-seq]

We disrupted the spatial Hh activation gradient in murine lung by inducing the constitutively active form of the Hh effector, Smo (SmoM2), throughout the entire GLI2+ stroma and performed RNA-seq on the bulk GLI2+ population. 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 bulk RNA-sequencing library was then subsequently generated. Cells were sequenced at a depth of average of 45 million reads/sample. The results reveal that expansion of Hh activation in the GLI2+ domain in the murine lung resulted in upregulation of genes that are expressed in the proximal stroma fibroblasts. Conversely, Hh activation downregulated genes enriched in the distal stroma as well as mesothelial cells. Four Gli2creERT2-tdt:R26RYFP/SmoM2 mice were administered intraperitoneally with tamoxifen for labelling the Hh-activated GLI2+ cells with yellow fluorescent protein (YFP) reporter expression, and four Gli2creERT2-tdt:R26RYFP mice were used as control. Whole adult murine lung tissue was dissociated to single cells, and subjected to FACS to select all live YFP+ cells. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. The GLI2+ cells were sorted by endogenous YFP fluorescence. Then the RNA of the samples were extracted and sent 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.