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
Riboseq-dweorg1 biol rep 2_s
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.