Enrichment and Sorting of Mammary Tumor Cells

Mammary tumors were dissociated into single cell suspensions through mechanical separation and enzymatic digestion as described^{22 (link)}. Dissociated tumor cells were enriched for Lin⁻ (CD45⁻/ CD31⁻/ TER119⁻/ BP-1⁻) mammary epithelial cells with StemCell Technologies EasySep Mouse Epithelial Cell Enrichment Kits per the manufacturer’s instructions. Lin⁻ cells were then incubated on ice for 20 min with anti-CD49f (α6 integrin) (BD Biosciences 555734) together with Alexafluor 647 (Invitrogen A21247) in PBS. Cells were spun down for 5 min at 550x g, then incubated with EpCAM-FITC conjugated antibody (Biolegend 118208) in PBS. Tumor cells were sorted on a BD FACS Aria cell sorter machine equipped with Diva software into their luminal (Lin⁻/ CD49f^low/EpCAM^high) and basal (Lin⁻/CD49f^high/EpCAM^low) subpopulations. Sorted cells were collected into 15ml conical tubes containing PBS. Genomic DNA was collected from sorted cell populations using Qiagen Blood and Tissue DNeasy spin column kit. Total RNA was collected from sorted cell populations using Qiagen RNeasy spin column kit. RNA was reversed transcribed using Invitrogen Superscript II First Strand Synthesis kit.