For the primary mammary gland scRNA-seq, Wild type (C57BL/6) mice were used. After preparation of primary mammary single cell suspension, we specifically sorted 2000 Procr+ MaSCs (CD31-, CD45-, Ter119-, CD24+, CD29hi) and also enrich 20000 mammary epithelial cells (CD31-, CD45-, Ter119-, CD24+), 30000 mammary immune cells (CD45+) and enrich 10000 stromal cells which contain endothelial cells and mesenchymal cells (CD45-, Ter119-, CD24-) and resuspended total cells at a density of 1000 cells per μl. And then 13,000 single cells were loaded for 10x single cell genomics RNA seq.
Filtering, alignment to the mm10 database and unique molecular identifier (UMI)-collapsing were performed using the Cell Ranger (v2) pipeline with default mapping arguments (10′ Genomics). Cell filtering: we used the Seurat 3 R package for data integration, analysis and visualization. To create Seurat object, genes which expressed in at least 3 cells and cells which had at least 200 detected genes, 1500 detected UMIs and no more than 5000 detected UMIs were selected. PC selection: the differentially expressed genes were found by “vst” method and the top 2000 differentially expressed genes were selected for PCA analysis. PCs selection was based on an elbow plot. 20 PCs were used for analysis. Dimensional reduction, cell clustering and data
Publications
Niche inflammatory signal regulates periodical mammary regeneration and safeguards stem cell survival under cytotoxic stress
