Summary:
Purpose: To comprehensively characterize VE-cadherin expressing lineages during adult skin homeostasis, to dissect their population-specific transcriptomic heterogeneity, and to investigate role of Alk1 in these linegease in vivo.Methods: We performed 10x genomics single-cell RNA sequencing (scRNA-seq) of FACS sorted tdTomato+ cells from tdTomato;Cdh5-CreERT2;Krt14-H2BGFP mice (for WT) or Alk1 flox flox;tdTomato;Cdh5-CreERT2 (For Alk1KO) back skin at indicated stage/genotype. FACS purified tdTomato+ single-cell suspension was processed for the barcoded single-cell 3â2 cDNA libraries generation using Chromium Single Cell 3â2 gel bead and library Kit v3. The final libraries were quantified using Agilent Bioanalyzer high sensitivity DNA chip and sequenced using an Illumina NextSeq-500. The raw data files were demultiplexed to generate the sample-specific FASTQ files, which were aligned to the mouse reference genome (mm10-2020-A) using the 10x Genomics Cell Ranger pipeline (v6.0.1). The raw scRNA-seq data was processed using Cell Ranger from the 10x platform to generate expression matrix for each sample that was further analyzed in R using the Seurat package version 4.0. Only high-quality cells that had between 200 and 5000 genes expressed and had under 10% of the UMIs mapped to mitochondrial genes were retained for further analysis.Results: We obtained more than 2000 high quality cells at each stage/genotype for further analysis by Seurat pipelineConclusions: Obtained high quality single cell transcriptomic data to dissect cellular and molecular heterogeneity of VE-cadherin expressing endothelial and non-endothelial lineages in adult mouse back skin.
