For scRNA-seq using the 10X Chromium system, FACS-sorted mammary basal and luminal cell populations were combined. Library generation and Illumina HiSeq-4000 sequencing were performed as previously described (Haensel et al., 2020 (link)). For scRNA-seq using the Fluidigm C1 platform, FACS-sorted basal MECs (Lin−CD49fhighEpCAM+) were washed and resuspended with Epicult-B medium (Stem Cell Technologies; Ca. No. 05610) at a concentration of ~500 cells/μL. Cell suspensions were mixed with Fluidigm C1 Suspension Reagents (Fluidigm 100–5315) at a ratio of 8:2 before loading onto the C1 chip (Fluidigm 100–5760). Bright-field images of captured cells were collected using a Keyence BZ-X710 microscope (Keyence Corporation, Itasca, Illinois, USA). Single-cell RNA isolation and amplification were performed using the Fluidigm C1 Single Cell Auto Prep IFC following the Fluidigm Protocol #100–7168 I1. RNA spike-in controls were omitted. cDNA library preparation was performed following the Fluidigm C1 Protocol #100–7168 I1.
Data analysis was performed using Seurat as we previously described (Haensel et al., 2020 (link)). Heatmaps were based on normalized, but not raw, values of expression to enable direct comparison across runs. A color gradient depicting the expression of each gene in each cell per average for all the cells was generated for each analysis combining biological replicates.
Data analysis was performed using Seurat as we previously described (Haensel et al., 2020 (link)). Heatmaps were based on normalized, but not raw, values of expression to enable direct comparison across runs. A color gradient depicting the expression of each gene in each cell per average for all the cells was generated for each analysis combining biological replicates.