Illumina HiSeq 2500 paired end sequencing; RNA-seq of human normal mammary gland cells FACS sorted into three populations: ALDH+, ALDH-CD44+CD24-, and ALDH-CD44-CD24+
RNA-seq of human normal mammary gland cells FACS sorted into three populations: ALDH+, ALDH-CD44+CD24-, and ALDH-CD44-CD24+
Normal (non-pathogenic) breast tissue was isolated from women undergoing voluntary reduction mammoplasty at the University of Michigan. The study protocol was approved by the University of Michigan Institutional Review Board.  Breast tissue was mechanically and enzymatically digested as previously described (Dontu et al., 2003; Kakarala et al., 2010). Normal mammary cells were stained for CD44, and CD24 expression and ALDH activity   accessed by the Aldefluor assay as described previously (Colacino et al., 2016) and analyzed or sorted on a Beckman Coulter MoFlo Astrios at the University of Michigan Flow Cytomtery core facility. Briefly, single mammary cells were first incubated with a lineage depletion cocktail that consisted of biotinylated antibodies targeted against CD45, HLA-DR, CD14, CD31, CD41, CD19, CD235a, CD56, CD3, CD16, and CD140b (all from eBioscience, except for CD140b (Biolegend) and CD41 (Acris)). Next, cells were stained with Alexafluor 750 tagged streptavidin, LIVE/DEAD Fixable Dead Cell Stain (Invitrogen), CD24 (Biolegend), CD44 (BD), and Aldefluor (Stem Cell Technology). Single color and isotype controls were included for compensation and gating purposes. Aldefluor-positive gating was based on DEAB (negative) controls. Flow cytometry data analysis was performed with FlowJo software v10.0.8. Total RNA was isolated from each cell population using the RNEasy Micro Kit (Qiagen) with on column DNase treatment. RNA concentration and quality was determined using a Nanodrop (Thermo) and Bioanalyzer (Agilent).  Ribosomal RNAs were depleted using Ribominus (Life) and sequencing libraries were prepared with the SMARTer Stranded RNA-Seq kit (Clontech).