GSM7566849: Ath RPFs, ligation-based Ribo-seq; Arabidopsis thaliana; OTHER
RPF isolation: RPFs were isolated as previously described (Trösch et al., 2018) with modifications described below. The units (U) of ribonuclease used in this study are normalized to one mL of plant lysate, derived from 100 mg of plant fresh weight. 300 mg frozen plant tissue was homogenized in liquid nitrogen with a mortar and pestle followed by the addition of 3 mL ribosome extraction buffer (0.2 M sucrose, 0.2 M KCl, 40 mM Tris-OAc pH 8.0, 10 mM MgCl2, 10 mM 2-Mercaptoethanol, 2% (v/v) polyoxyethylene (10) tridecyl ether, 1% (v/v) Triton X-100, 100 μg/mL chloramphenicol, 100 μg/mL cycloheximide). After brief mixing, a 0.5 mL aliquot of the lysate was flash frozen and stored at -80 °C for later total RNA extraction using TRIzol reagent (ThermoFisher cat# 15596026). This total RNA can be used for standard RNA-seq experiments. This step is especially important if the calculation of translation efficiencies is desired (i.e., the normalization of Ribo-seq data to RNA-seq data). The remaining lysate was filtered through glass wool, followed by centrifugation for 10 min at 15,000 x g at 4 °C to remove cell debris. 2.5 mL of the clarified lysate were incubated at specified concentrations of RNase1 or MNase for 1 h at room temperature with gentle rotation to degrade mRNA regions that are not covered and protected by translating ribosomes. Since Ca2+ is a known cofactor of MNase, samples digested with MNase include 5 mM CaCl2. The ribonuclease-treated lysate contains mainly monosomes which were loaded onto a 2 mL sucrose cushion (30% (w/v) sucrose, 40 mM Tris-Acetate pH 8.0, 100 mM KCl, 15 mM MgCl2, 0.1 mg/mL chloramphenicol, 0.1 mg/mL cycloheximide, 0.2% β-MeEtOH) and centrifuged (OptimaTM L-80 XP Ultracentrifuge - Beckman Coulter) for 1.5 h at 303,800 x g at 4 °C. The supernatant was then carefully aspirated and the pelleted monosomes resuspended in 0.5 mL footprint isolation buffer (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0, 100 mM NaCl, 1% (w/v) SDS, 0.1 M EGTA pH 8.0). The RNA was immediately extracted from this pellet using 0.5 mL TRIzol reagent. Next RPFs were size-selected through electrophoresis on a 12% denaturing polyacrylamide gel (19:1, acrylamide:bisacrylamide) prepared in 1x TBE buffer (89 mM Tris, 89 mM Boric Acid, 2mM EDTA pH 8.0) containing 8 M urea. To this end, 25 μg RNA were resuspended in 40 μL of ribosome footprint loading buffer (90% (v/v) deionized formamide, 20 mM Tris-HCl pH 7.5, 20 mM EDTA pH 8.0, 0.04% (w/v) bromophenol blue, and 0.04% (w/v) xylene cyanol) and denatured for 10 min at 70 °C. The gel was run in 1x TBE buffer with a constant power of 30 W at constant temperature of 12 °C (achieved by a cooling unit). Co-migrating pre-stained RNA ladder (Biodynamics Laboratory cat# DM253) was used to visualize the regions of the gel to excise RPFs. All RPFs that were not rRNA-depleted were size-selected between 20-50 nt. All rRNA-depleted RPFs were size-selected between 20-35 nt. RNA was eluted from the excised gel piece in 4 mL TESS (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0, 0.1 M NaCl, 0.2% (w/v) SDS) by overnight incubation at 4 °C with gentle rotation. Eluted RNA was isolated with 4 mL of phenol:chloroform:isoamyl alcohol (25:24:1), followed by overnight ethanol precipitation at -20 °C. To increase purity and further narrow the volume, the RNA pellet was resuspended in 0.1 M NaCl (500 μL) and subjected to a second round of phenol:chloroform:isoamyl alcohol (25:24:1) extraction, followed by two washes with chloroform:isoamylalcohol (24:1) and overnight ethanol precipitation at -20 °C. The received RPF pellet was washed twice with 75% ethanol and resuspended in 20 μL RNase-free water. For the ligation-free strategy, RPFs were directly used as input for the D-plex small RNA-seq kit (Diagenode cat#C05030001), according to manufacturer's instructions. For the RNA-ligase strategy, the terminal ends of the RPFs were first repaired using T4 polynucleotide kinase, and used as input into the NEXTflex small RNA-seq kit V4 (Perkin Elmer, cat#NOVA-5132-31), according to the manufacturer's instructions.