Rnaeasy microrna extraction kit

Myc-DDK-tagged mouse ribosomal protein L22 like 1 (Rpl22l1) cDNA was purchased from Origene (MR200606 representing NM_026517) and packaged into AAV9-EF1a-DIO-Rpl22l1-Myc-DDK-2A-tdTomato-WPRE by Virovek with titres of 2.24 × 10¹³ viral genomes per ml. AAV viral vector carrying RiboTag was stereotaxically injected into the SNc in Dat-cre mice and cNdufs2^−/− mice. Four weeks after the injection, the mice were euthanized and the SNc tissue expressing RiboTag was dissected and placed at −80 °C. RiboTag immunoprecipitation was performed as described previously^{45 (link)}. In brief, tissue was homogenized in cold homogenization buffer (50 mM Tris (pH 7.4), 100 mM KCl, 10 mM MgCl₂, 1 mM dithiothreitol, 100 μg ml⁻¹ cycloheximide, protease inhibitors and recombinant RNase inhibitors, and 1% NP-40). Homogenates were centrifuged 10,000g for 10 min, and the resulting supernatant was precleared with protein G magnetic beads (Thermo Fisher Scientific) for 1 h at 4 °C with constant rotation. Immunoprecipitations were carried out with anti-Flag magnetic beads (Sigma-Aldrich) at 4 °C overnight with constant rotation. Four washes were carried out with high-salt buffer (50 mM Tris (pH 7.4), 350 mM KCl, 10 mM MgCl₂, 1% NP-40, 1 mM dithiothreitol and 100 μg ml⁻¹ cycloheximide). RNA extraction was performed using the RNA-easy Micro RNA extraction kit (QIAGEN) according to the manufacturer instructions.