GPAT5_R1_1
Arabidopsis thaliana
Protocols: Plants have been sampled 8DAG, after growth on control medium and under continuous light. To restrict the analysis to the peridermis (avoiding the endodermis where GPAT5 is also being expressed in young parts of the root,), only a section of the root comprised between the root-hypocotyl junction and the first emerged lateral root was manually dissected. Seedlings were sown on solid half MS medium (Duchefa Biochemie B.V.) supplemented with 1% of Sucrose (VWR), 0.1g/L Myo-inositol (Sigma-Aldrich), 0.5 g/L MES (Duchefa Biochemie B.V.) and 0.8% Plant Agar (Lab M, MC029). Plates were stratified for 2 days at 4°C and grown at 21°C under continuous light conditions. For RNA extraction frozen plant powder was homogenized in ice-cold polysome extraction buffer (200 mM Tris-HCl (pH = 9.0), 200 mM KCl, 36 mM MgCl2, 25 mM ethylene glycol tetraacetic acid, 1 mM dithiothreitol, 50 μg/ml cycloheximide, 50 μg/ml chloramphenicol, 1% Igepal CA-630, 1% Brig-35, 1% Triton X-100, 1% Tween-20, 2% polyoxyethylene (10) tridecyl ether, 1% sodium deoxycholate). After incubation for 10 min on ice, homogenates were centrifuged at 16,000 × g and 4 °C for 10 min to pellet insoluble cell debris. Affinity purification of FLAG-RPL18-containing polysomes was carried out using anti-FLAG beads (Sigma-Aldrich) incubated with the supernatant at 4 °C for 3h. Gels were subsequently collected by centrifugation, and washed four times with polysome wash buffer (200 mM Tris-HCl (pH = 9.0), 200 mM KCl, 36 mM MgCl2, 25 mM ethylene glycol tetraacetic acid, 5 mM dithiothreitol, 50 μg/ml cycloheximide, 50 μg/ml chloramphenicol). Polysomes were eluted by resuspension of the washed gel in wash buffer containing 3× FLAG peptide. Total RNA was extracted from the final elution using Direct-zol RNA kit (Zymo research). DNAse treatment was performed in column according the manufacturer instructions. Per sample, an amount of 1ng of total RNA was used as input for the SMART-Seq v4 Ultra Low Input RNA protocol (version \091817\) from Takara Bio USA, Inc. Subsequently, 1 ng of purified cDNA was sheared to 300 bp using the Covaris M220. From the sheared material, sequencing libraries were prepared with the NEBNext Ultra DNA Library Prep Kit for Illumina (version 7.0 - 3/18), according to the manufacturer's protocol including a size selection to 250 bp insert size.