Sample 5
Arabidopsis thaliana
Protocols: Mitochondria were collected from two-week-old in vitro grown plants in shaking flasks. Plants were grown in soil-filled pots in long day conditions (16h light) at approximately 120 umol s-1 m-2. RNA immunoprecipitation (RIP) assays were performed with the μMACS GFP-Tagged Protein Isolation Kit (Miltenyi Biotec) according to manufacturer's instructions with minor modifications. Samples were homogenized in RIP lysis buffer (20 mM HEPES-KOH, pH 7.6, 100 mM KCl, 20 mM MgCl2, 1 mM DTT, 1% (v/v) Triton X-100, 1X of complete EDTA-free protease inhibitor (Roche)) for 30 min at 4°C with slow rotation (10 rpm). The lysates were clarified by centrifugation at 100,000 g for 20 min at 4°C and resulting the supernatant was incubated with 50 μL of anti-GFP magnetic beads (Miltenyi Biotec) for 30 min at 4°C with rotation (10 rpm). Following three washes with RIP washing buffer (lysis buffer with 0.1% Triton X-100) and recovery of the beads on a magnetic stand each time, RNAs were extracted from the beads with TRIzol reagent (Life Technologies), precipitated with ethanol, and then used for RT-qPCR analysis. Prior to retrotranscription, immunoprecipitated RNAs were treated with DNase Max (QIAGEN). Ribosome footprints were depleted from ribosomal RNAs after first strand cDNA synthesis using custom biotinylated oligonucleotides matching major rRNA contaminants found in previous experiments (Planchard et al., 2018).N. Planchard, P. Bertin, M. Quadrado, C. Dargel-Graffin, I. Hatin, O. Namy, H. Mireau, The translational landscape of Arabidopsis mitochondria. Nucleic acids research 46, 6218-6228 (2018).