GSM3042965: MCF7_RNAseq; Homo sapiens; RNA-Seq
Total RNA was extracted from cytoplasmic lysates with acid-phenol:chloroform:isoamylalcohol and isopropanol precipitation and ribosomal RNA was removed with Ribo-Zero rRNA Removal Kit (Illumina): Libraries were prepared using the TruSeq RNA Library Prep Kit (Illumina) following the manufacturer's instructions. Cells (1.5 x 10^6) were seeded in 10 cm cell culture dishes and kept in culture until reaching 80% confluency. Prior to lysis, cycloheximide (10 μg/mL) was added to the culture that was incubated in this condition at 37°C for 4 min. The dishes were then rinsed with ice cold PBS supplemented with 10 μg/ml cycloheximide and lysated with 150 μl of polysomes lysis buffer (10 mM NaCl, 10 mM MgCl2, 10 mM, TRIS/HCl-pH 7.5, 1% Triton X-100, 2 U/μl RNAse inhibitor, 1M DTT, 1% Na deoxycholate). The lysates were incubated for 5' in ice, vortexed and centrifuged for 5' at 14.000rpm at 4°C to precipitate cellular debris, nuclei and mitochondria. The supernatants have been stored at -80°C before the libraries preparation.