Illumina HiSeq 2500 sequencing; RNA-seq of two human mammary epithelial cell lines (HMLE and HMLE-Twist-ER) treated with 4-hydroxytamoxifen vs control
RNA-seq of two human mammary epithelial cell lines (HMLE and HMLE-Twist-ER) treated with 4-hydroxytamoxifen vs control
For live cell FACS sorting, cells were washed once with pre-warmed PBS, incubated for 5 min at 37 °C with pre-warmed TrypLE 1X Express (Gibco), pipetted off the cell culture dish, and collected in 4 °C PBS. Cells were pelleted at 350 x g for 5 min at 4 °C, re-suspended in 4 °C PBS with 1% BSA, and stained with E-Cadherin-AF647 (clone 67A4, 5 µg/100µl, Biolegend) and CD44-PE (clone IM7, 1.25 µg/100 µl, Biolegend) for 20 min at 4 °C in the dark. Cells were washed once using PBS with 1% BSA and kept on ice until FACS sorting using the FACSAria III (BD Biosciences).  The HMLE and HMLE-Twist-ER (HTER) cell lines were a gift from the laboratory of Prof. Robert Weinberg at the Whitehead Institute for Biomedical Research and the Massachusetts Institute of Technology and were cultured in a 1:1 mixture of DMEM F12 Ham medium (Sigma Aldrich) supplemented with 10 µg/ml human insulin (Sigma Aldrich), 10 ng/ml EGF (Peprotech), 500 ng/ml hydrocortisone (Sigma Aldrich), and PenStrep (Gibco) with the mammary epithelial growth medium (MEGM) BulletKit (Lonza). For HTER cells, the growth medium was supplemented with 1 µg/ml Blasticidin S (InvivoGen). For RNA isolation, cells were pelleted at 350 x g for 5 min at 4 °C and lysed in 350 µl RLT buffer of the RNeasy Mini Kit (Qiagen). RNA was isolated according to the manufacturer's instructions. Briefly, RNA was collected on the RNeasy spin column, washed with 70% ethanol (Merck), and DNA was removed by incubation with DNAse I (Qiagen). RNA was collected in 30-50 µl diethylpyrocarbonate (DEPC, Sigma Aldrich)-containing water and stored at -80 °C. DEPC water was prepared by dissolving 1 ml DEPC in 1 L ddH2O prior to autoclavation. The RNA quality was assessed using a NanoDrop (Thermo Scientific) and Bioanalyzer (Agilent).  Library construction was performed using poly(A) enrichment and the Illumina TruSeq stranded protocol