GSM6165846: upf1-2; Saccharomyces cerevisiae; RNA-Seq
For each sample, 250 ml of cells were harvested by centrifugation at room temperature and directly frozen in liquid nitrogen after supernatant removal. Cells were lysed in 1X lysis buffer (10 mM Tris-HCl pH 7.4, 100 mM NaCl, 30 mM MgCl2) supplemented by 2X cOmplete Protease Inhibitor Cocktail (Roche) and ribosome protected fragments (RPFs) were prepared as previously described (Baudin-Baillieu et al., 2016. Methods Mol Biol), with the following modifications. Polysomes were purified on sucrose cushion and then digested with Rnase I (Ambion, 5 units/UA260). Ribo-depletion was performed using in-house designed oligonucleotides. Libraries were then prepared from 10 ng of RPFs using the D-Plex Small RNA-Seq kit for Illumina (Diagenode) and the D-Plex Unique Dual Indexes for Illumina – set A (Diagenode).