Harringtonine

Cells were seeded at 1 × 10⁶ cells per 10-cm culture dish and cultured overnight. MCF10A-ER-Src cells were treated by 1 µM 4-hydroxy-tamoxifen for various time points (1, 4, and 24 hr) to induce transformation. Cells were pretreated with cycloheximide (100 µg/ml; Sigma-Aldrich, St. Louis, MO) for 90 s or harringtonine (2 µg/ml; Santa Cruz, Santa Cruz, CA) for 5 min, and detergent lysis was then performed with flash-freezing in liquid nitrogen. For ribosome profiling, DNase I-treated lysates were then treated with RNase I, and ribosome-protected fragments were purified for Illumina TruSeq library construction as previously described (Ingolia et al., 2012 (link)). For RNA-seq, total RNA was purified from DNase-treated lysates, and ribosomal RNA was depleted with RiboMinus Eukaryote Kit (Thermo Fisher Scientific, Waltham, MA). RNA-seq libraries were prepared with a tagging-based workflow (Pease and Kinross, 2013 ). In brief, rRNA-depleted RNA was fragmented at 85°C for 5 min, followed by cDNA synthesis, terminal tagging and PCR amplification with ScriptSeq v2 RNA-Seq Library Preparation Kit (Epicentre, Madison, WI). Ribosome profiling and RNA-seq libraries were sequenced with Illumina HiSeq 2500.