The m6A demethylase FTO promotes C/EBPβ-LIP translation to perform oncogenic functions in breast cancer cells (Ribosome profiling)
The m6A demethylase FTO promotes C/EBPβ-LIP translation to perform oncogenic functions in breast cancer cells (Ribosome profiling)
Cells were grown as usual and harvested at 80% confluency in ice cold PBS with 100 μg/ml cycloheximide. Cells were lysed in lysis buffer and lysates were treated with RNase I before ultracentrifugation over a sucrose gradient. Cytosolic ribosome fractions were collected and digested with proteinase K in the presence of 1% SDS prior to RNA isolation using TRIzol. RNA fragments were run on a denaturing urea polyacrylamide gel and correctly sized fragments were eluted and recovered by ethanol precipitation. RNA was 3' dephosphorylated and preadenylated linkers were ligated to the fragments. Following 5' deadenylation and RecJ exonuclase treatment, fragments were cleaned using an Oligo Clean & Concentrator kit and rRNA was depleted by substractive hybridization using streptavidin beads and biotinylated rRNA oligos. Following ethanol precipitation, samples were reverse transcribed and again ethanol precipitated prior to gel purification using urea PAGE. Fragments were circularized, recovered by ethanol precipitation and subjected to library construction PCR. Size selection was performed using nondenaturing PAGE and fragments were recovered by ethanol precipitation. Library quality was confirmed using a Bioanalyzer 2100 (Agilent) with the DNA 7500 kit (Agilent) and libraries were quantified by Qubit dsDNA BR assay (ThermoFisher).