Differential RNA-seq expression analysis of sorted subpopulations and mammospheres and pooled shRNA screening strategy. (A) Flow cytometric profiles of Lin− mammary cell populations showing representative sort gates for CD29hiCD24+ MaSC-enriched basal cells (MaSC/basal), CD29loCD24+CD61+ luminal progenitor-enriched cells (LP) and CD29loCD24+CD61− mature luminal cell-enriched cells (ML). (B) Multidimensional scaling plot of expression data generated by RNA-Seq of populations including MaSC/basal (Basal), LP and ML populations, MaSC/basal-derived mammospheres (Mammosphere) and Comma-Dβ cells (CommaDβ). (C) Schematic outline of the screening strategy. (D) Plot showing the relative frequency of shRNA-carrying cells at T2 and T14 as determined by next-generation sequencing, including a non-silencing control hairpin (non-sil). Dashed lines, 1.5-fold change. (E) Numerical summary of shRNA performance in the screen.