title: GSM3863557: WT_Total; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: Mitochondria were isolated from leaves as described by Lister et al. (2007). Approximately 15 g (fresh weight) of leaves from rps10 and 30 g from wild-type plants were used per one isolation, yielded typically ~1 mg of mitochondrial proteins. Mitochondrial fractions were frozen in liquid nitrogen and stored at -800C. Mitochondrial Ribo-Seq and RNA-Seq libraries were prepared using the ARTseq Ribosome Profiling Kit (Epicentre) dedicated for yeast, with slight modifications. Each library was prepared from fifteen independent mitochondrial preparations. Immediately after removing mitochondrial preparations from the -80°C freezer chloramphenicol was added to a final concentration of 100 μg/ml and thawed mitochondria were pooled, suspended in polysome extraction buffer (0.2 M sucrose, 0.2 M KCl, 10 mM MgCl2, 2% polyoxyethylene (10) tridecyl ether, 1 mM DTT, 1% Triton X-100, DNase I (0.01 U/μl), 100 μg/ml chloramphenicol, 40 mM Tris-acetate, pH 8.0), incubated on ice for 10 minutes and centrifuged for 10 minutes at 28,154 x g at 4°C to obtain clarified lysate. RNA concentration in the lysate was determined as OD260 and aliquots of 100 (mtRNA-seq library) and 200 μl (mtRibo-Seq library) were prepared. The 100-μl aliquot was supplemented with 10 μl of 10% SDS and total RNA was purified using the RNA Clean & Concentrator-25 kit according to the protocol for large RNAs >200 nt (Zymo Research). The 200-μl aliquot was treated with ARTseq nuclease (0.3 U nuclease per 1 OD260 of lysate) to degrade unprotected RNA following Epicentre instruction, and ribosome protected fragments (RPFs) were isolated using sucrose cushion ultracentrifugation at 4°C for 4 hours at 294,000 x g. The pellet was suspended in 100 μl nuclease-free water and 10 μl of 10% (w/v) SDS was added. Next, the RPFs were purified using RNA Clean & Concentrator-25 kit according to the protocol for small RNAs (17-200 nt). After purification the total RNA and RPFs preparations were depleted of rRNA using the Ribo-Zero rRNA Removal Plant Leaf kit (Illumina) and then were purified again using the RNA Clean & Concentrator-25 kit according to the protocol for large RNAs >200 nt for total RNA and the RNA Clean & Concentrator-5 kit according to the protocol for RNA >17 nt for RPFs. The total RNA preparation without ribosomal RNA was heat-fragmented. The RPFs without ribosomal RNA were purified by 15% TBE-urea PAGE (Thermo Fisher Scientific) and the gel fragment containing RNA of 25 to 35 nt was excised. The RPFs were recovered from the excised gel slices following overnight elution as specified in the ARTseq Ribosome Profiling Kit. The fragmented total RNA and RPFs were end-polished, purified by RNA Clean & Concentrator-5 kit according to protocol for >17 nt, ligated with 3' adaptors, reverse-transcribed and purified as above. Purified cDNAs were separated on 10% TBE-urea PAGE, and gel fragments containing ssDNA of 80 to 100 nt for cDNA generated from total RNA fragments, and 70 to 80 nt for cDNA from RPFs, were excised. cDNA was recovered by overnight elution as specified in the ARTseq Ribosome Profiling Kit. The purified cDNA was then circularized and used for PCR with index PCR primers for 9 cycles. The libraries were purified by 8% Native TBE PAGE and AMPure XP beads (Beckman Coulter) and their quality and concentration was determined using an Agilent 2100 Bioanalyzer. High-throughput sequencing was performed on an Illumina Miseq 2500 sequencer in the single-end mode for 50 nucleotides at Wyzer Biosciences (Cambridge, MA, USA)