NextSeq 500 sequencing; Ribosome profiling in mitochondria from flowers of wildtype Arabidopsis thaliana and DWEORG1 (At3g15590) mutant
Ribosome profiling in mitochondria from flowers of wildtype Arabidopsis thaliana and DWEORG1 (At3g15590) mutant
Flowers were harvested simultaneously for all genotypes, snap-frozen in liquid nitrogen and stored at -80°C until use. The plants were grown in the Greenhouse under long day conditions.  Total ribosome footprints were prepared from a mix of flower buds and open flowers. About 2.5 g of flowers were ground in liquid nitrogen and resuspended in 15 ml of ribosome extraction buffer (0.2 M sucrose, 0.2 M KCl, 40 mM Tris-acetate, pH 8, 10 mM MgCl2, 10 mM b-mercaptoethanol, 2% polyoxyethylene 10 tridecyl ether, 1% Triton X-100, 100 mg/ml chloramphenicol and 100 mg/ml cycloheximide). The obtained solution was centrifuged twice for 10 min at 15 000 g at 4◦C to remove the cell debris and the supernatant was transferred to a new tube. To estimate the RNA concentration contained in the supernatant, total RNA was extracted from a 0.5 ml aliquot using the TRI Reagent® (Sigma- Aldrich) following the manufacturer's instructions. The supernatant was then digested with 10 U/OD260nm/ml of RNAse I (Ambion), and incubated 1 h at 25◦C to degrade unprotected RNA. The solution was then layered on an equal volume of a sucrose cushion (1 M Sucrose, 0.1 M KCl, 40 mM Tris-acetate, pH 8, 15 mM MgCl2, 5 mM b-mercaptoethanol, 100 mg/ml chloramphenicol and 100 mg/ml cycloheximide) and centrifuged for 3 h at 55 000 g at 4◦C in a TFT 65-13 rotor (Kontron). The obtained pellet was resuspended in 0.6 ml of RNA extraction buffer I (0.1 M EGTA pH 8, 0.1 M NaCl, 1% sodium dodecyl sulphate (SDS), 10 mM Tris–HCl pH 8 and 1 mM ethylenediaminetetraacetic acid (EDTA)), and RNAs were extracted with the TRI Reagent® (Sigma-Aldrich) following the manufacturer's recommendations. Ribosomal footprints were further purified on 17% (v/v) acrylamide- bisacrylamide (19:1) gel made with 7 M urea and 1 × Tris- acetate-EDTA (TAE) buffer. For this, 600 mg of RNAs were electrophoresed in two separated 15 cm-wide wells. The gel was pre-run for 1 h at 50◦C and then run for 15–16 h at 90 V at the same temperature. A mix comprising 100 ng of 27- mer and 33-mer RNA oligonucleotides was loaded on both gel extremities and used as size marker. After staining for 30 min in 1× SYBR Gold (Life Technologies) diluted in 1× TAE, RNA contained in the 27 and 33 nt regions were separately excised from the gel. The gels slices were stored at least 2 h at -20◦C and then physically disrupted by centrifugation through a needle hole made in a 0.5 ml microcentrifuge tube nested in an outer 1.5 ml collection microtube. The tube assembly was centrifuged at maximum speed for 5 min. RNAs were eluted overnight from gel fragments by passive diffusion in 5 ml of RNA extraction buffer II (10 mM Tris–HCl pH 8, 1 mM EDTA, 0.1 M NaCl, and 0.2%SDS) at 4◦C on a rotating wheel. The recovered RNAs were then isolated with the TRI Reagent® (Sigma-Aldrich) following the manufacturer's instructions, resuspended in 30 ml of RNAse-free water and stored at −80◦C until use. Ribosome footprints were depleted from rRNA with the RiboMinusTM Plant Kit for RNA-Seq (invitrogen), following the manufacturer's instructions. The sequencing libraries of ribosomal footprints of two biological repeats per genotype were prepared using the NEXTflex Small RNA-Seq Kit v3 (Bioo Scientific) for Ribo-Seq following the manufacturer's instructions.