GSM3042964: MCF7_RIBOseq; Homo sapiens; OTHER
Cytoplasmic lysates were digested with 5 Units of ARTseq Nuclease for each A260 of lysate at room temperature for 45 minutes. The RNA was treated with Ribo-Zero rRNA Removal Kit (Illumina) to remove ribosomal RNA contamination and the Ribosome Protected Fragments (RPFs) were then extracted with acid-phenol:chloroform:isoamylalcohol and isopropanol precipitation and size seNGS libraries were prepared starting from RPFs using the ARTseq Ribosome Profiling Kit – Mammalian (Epicentre) following the standard protocol. 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.