GSM5596417: RNA_Sminus_rep1 [S10_rep1]; Sorghum bicolor; RNA-Seq
A whole shoot (2 g) was frozen in liquid nitrogen and powdered in a Multi-beads Shocker (Yasui Kikai, Osaka, Japan). Approximately 100 mg of powder was used for RNA-seq library construction,  and the rest was used for Ribo-seq. For Ribo-seq, the powder was homogenized  in ice-cold polysome extraction buffer [100 mM Tris·HCl (pH 8.0), 40 mM KCl, 20 mM MgCl2, 1 mM dithiothreitol, 100 µg/mL cycloheximide, 2% polyoxyethylene (10) tridecyl ether, 1% deoxycholic acid, 10 U/mL DNase I (Qiagen, Hilden, Germany)]  in a Dounce homogenizer and incubated on ice for 10 min. The homogenates were centrifuged for 30 min at 16,000× g and 4 °C in a JA25.50 rotor (Beckman Coulter,  CA, USA) to pellet insoluble cell debris. Each supernatant was layered on the top of a 1.75-M sucrose cushion [100 mM Tris·HCl (pH 8.0), 40 mM KCl, 20 mM MgCl2, 1 mM dithiothreitol, 100 µg/mL cycloheximide, 1.75 M sucrose] and centrifuged for 18 h at 170,000× g and 4 °C in a TY70 Ti  rotor (Beckman Coulter). The polysome pellet was resuspended in the same buffer without sucrose . The OD260 was determined with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific,  MA, USA); 1 OD260 unit of polysomes was digested with 20 units of Epicentre RNase I (Lucigen, WI, USA) in a 90-mL reaction mixture for 45 min at 25 °C,  and 10 mL of SUPERase-In RNase inhibitor (Thermo Fisher Scientific) was added. Monosomes were isolated on Illustra MicroSpin S-400 HR columns (GE Healthcare,  IL, USA). RNA was extracted and concentrated with an RNA Clean & Concentrator Kit (Zymo Research,  CA, USA). Concentrated RNA was separated by 7 M urea–12% PAGE in TBE buffer [89 mM Tris·HCl (pH 8.0), 89 mM boric acid, 2 mM EDTA]. Gel slices corresponding to the 26–34-nt region were excised, and a ribosome profiling library was prepared according to McGlincy and Ingolia 2017 with several modifications. To extract RNA or reverse transcription products, crushed gel pieces were resuspended in 45 mM ammonium acetate, 0.045% SDS. For rRNA depletion, a Ribo-Zero Plant Leaf Kit (Illumina,  CA, USA) was used in ¼-scale reactions.   For RNA-seq, total RNA was extracted from approximately 100 mg of frozen tissue powder of the whole shoot using Trizol Reagent (Thermo Fisher Scientific). DNA was depleted with a Turbo DNA-free Kit (Thermo Fisher Scientific), and RNA was purified with an RNA Clean and Concentrator Kit-5 (Zymo). Poly-(A) mRNA was enriched using a NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, MA, USA). A library for Illumina sequencing was constructed using a NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs).