GSM5900265: riboRNAseq_KO3F; Mus musculus; RNA-Seq
To prepare ribosome profiling libraries, snap-frozen cerebellar tissue (rapidly dissected on ice) was homogenized in Polysome buffer (20 mM Tris·Cl (pH 7.4), 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 µg/ml Cycloheximide, 1% Triton X-100, 25 U/ml DNase I). A fraction of the lysate was digested with RNAse I (Ambion; 100U/μL) (footprints) and purified through a MicroSpin S-400 column (GE Healthcare) to size-select for monosomes, while the remaining fraction was kept as an internal mRNA control (total RNA; RNA sequencing). All samples were depleted of ribosomal RNA using the Ribo-Zero Gold (Human/Mouse/Rat) Kit (Illumina). The footprint samples were size-selected for bands running between 28 and 34 nucleotides on a 15% TBE-Urea polyacrylamide gel (ThermoFisher Scientific), while total RNA samples were heat fragmented by incubating at 95 °C in an alkaline fragmentation solution (50 mM NaCO3 pH 9.2, 2 mM EDTA) for 40 min, yielding comparably sized fragments to footprints. The quality and concentration of samples was assessed by running an Agilent Small RNA chip (Agilent Technologies). sequencing libraries were generated using the NEXTflexTM Small RNA Sequencing Kit v3, according to the manufacturer's instructions