title: RNAseq of MCF7 cell line: polysomal RNA
design: MCF7 cells were cultured in RPMI (Gibco, Life Technologies, Milan, Italy) supplemented with 10% FBS, antibiotics (100 units/ml penicillin plus 100 mg/ml streptomycin), and 2mM L-glutamine. Polysomal profiling was performed after 16-hour treatments by 1 M doxorubicin, 10M Nutlin, or DMSO control. Cytoplasmic lysates were prepared, loaded in 1550% linear sucrose gradients, ultra-centrifuged and fractionated with a Teledyne fraction collector as recently described. All fractions containing sub-polysomal or polysomal RNA were identified from the absorbance profile and pooled into two separate 15 ml falcon tubes. RNA was purified by adding one volume of acid phenol: chloroform: isoamyl alcohol (pH 4.5) followed by isopropanol precipitation and a wash step in ice-cold 70% ethanol. Total RNA was also extracted using the Agilent Total RNA Isolation Mini Kit (Agilent Technologies, Milan, Italy). Sequencing libraries were prepared following the manufacturer instructions and the TruSeq RNA Library preparation kit. We used 1 g of RNA as input and assessed the input RNA quality (RNA Integrity Number, RIN >8) using the Agilent RNA 6000 Nano kit on an Agilent 2100 Bioanalyzer. Two replicates were generated for each condition. The final libraries were run on a 2100 Bioanalyzer (Agilent) using the Agilent DNA 1000 Kit (Agilent #5067-1504) and assayed on the Qubit fluorometer using the Qubit dsDNA HS Assay Kit (Thermo Scientific #Q32851) for quality check and quantification. Libraries were then sequenced on an Illumina HiSeq2504