Phenotypically supervised single cell sequencing parses within-cell-type heterogeneity

We used a phenotypic cell sorting technique to ask whether phenotypically supervised scRNAseq analysis (pheno-scRNAseq) can provide more insight into heterogeneous cell behaviors than unsupervised scRNAseq. Using a simple 3D in vitro breast cancer (BRCA) model, we conducted pheno-scRNAseq on invasive and non-invasive cells and compared the results to phenotype-agnostic scRNAseq analysis. Pheno-scRNAseq identified unique and more selective differentially expressed genes (DEGs) than unsupervised scRNAseq analysis. Functional studies validated the utility of pheno-scRNAseq in understanding within-cell-type functional heterogeneity and revealed that migration phenotypes were coordinated with specific metabolic, proliferation, stress, and immune phenotypes. Transcriptomic examination of human and mouse breast cancer cells for phenotypic heterogeneity. Phenotypic supervision: Cells were phototagged using photoconversion of Dendra2 that was transduced into MDA-MB-231 cells. Cells were then sorted using fluorescence activated cell sorting. 2 samples were phenotypically supervised, 2 samples were phenotype-agnostic.