RNA-seq analysis of MCF-10A human mammary epithelial cells following MYC activation

We profiled gene expression and splicing changes in MCF-10A human mammary epithelial cells expressing MYC fused to a portion of estrogen receptor (MYC-ER) (Eilers et al., 1989; Littlewood et al., 1995). We performed RNA-seq, in triplicate, on 3D-grown MCF-10A MYC-ER cells at 0, 8, and 24 hours (h) after 4-OHT-induced MYC activation. As a control for 4-OHT-induced effects, 3D-grown parental MCF-10A cells lacking the MYC-ER fusion protein were treated with 4-OHT at the same timepoints. Overall design: For 3D culture assays, MCF-10A and MCF-10A MYC-ER cells were seeded at a density of 10,000 cells per well in triplicate on a 4-well glass chamber slide coated with Matrigel Growth Factor Reduced (BD Biosciences) as described (Debnath et al., 2003; Liberzon et al., 2015). Media was replaced at 72h. Starting on day 3, cells were treated with 1µM 4-hydroxy tamoxifen (4-OHT) (Sigma) for 48h, 24h, 8h, or 0h, prior to collection. All samples were collected at the same time on day 5. Cells were washed with PBS (1X) and the Matrigel was dissolved by incubating slides at 4°C in Cell Recovery Solution (BD Biosciences). Total RNA was extracted using the RNeasy kit (Qiagen) including DNase I treatment per manufacturer instructions. Barcoded RNA libraries were prepared starting with 1ug for MCF-10A of total RNA using the TrueSeq stranded mRNA kit with polyA selection (Illumina), and quantified using a Bioanalyzer DNA 1000 chip (Agilent). Libraries were sequenced as 150bp paired-end reads at 100-200 million reads per library on an Illumina HiSeq, equal amounts of 3 libraries were pooled per lane.