title: GSM6940580: h32d2d4 Ribo seq2; Arabidopsis thaliana; OTHER
library_name: GSM6940580
library_construction_protocol: 0.2g of inflorescence tissue was ground into fine powder in liquid nitrogen and the powder was lysed in 0.45ml Buffer D, which consists of 100 mM Tris-HCl pH8.0, 40 mM KCl, 20 mM MgCl2, 2% (v/v) polyoxyethylene (10) tridecyl ether (Sigma-Aldrich, P2393), 0.5% (w/v) sodium deoxycholate (Sigma-Aldrich, D6750), 1mM dithiothreitol (Thermo-Fisher, R0861), 100 ug/ml cycloheximide (Sigma-Aldrich, C1988) and 10 U/ml DNase I (Roche, 4716728001). After 10 minutes incubation on ice, two rounds of centrifugation at 14,000 rpm for 10 minutes at 4°C were applied to generate cleared supernatant marked as total lysate. The OD260 of the cleared total lysate was measured by a NanoDrop spectrophotometer (Thermo Scientific, ND-2000), using Buffer D as the blank. Around 2,000U OD260 of the total lysate was treated with 75U RNase I (Thermo Fisher, AM2294) at 23°C for 1 hour. The reaction was stopped by adding 300U SUPERase•In RNase Inhibitor (Thermo Fisher, AM2694) and further processed with Amersham MicroSpin S-400 HR columns (Cytiva, 27514001). Ribosome protected footprints (RPFs) were isolated and purified using the RNA Clean & Concentrator-5 kit (Zymo Research, R1016). Later, rRNA depletion was performed as described before 19, using Dynabeads MyOne Streptavidin C1 (Thermo Fisher, 65001) and custom-made biotinylated DNA oligonucleotides listed in Table S1. The rRNA-depleted RPFs were concentrated by the RNA Clean & Concentrator-5 kit and fragments of 20-nt to 40-nt were selected from a 15% urea-PAGE gel. Sliced gel pieces were ground by RNase-free 1ml Olympus Premium Reach Pipet Tips (Genesee Scientific, 23-165RL). Extraction buffer (0.5M ammonium acetate, 10% SDS) was added to extract RPFs at room temperature for 2 hours. Gel debris was removed by Corning Costar Spin-X centrifuge tube filters (Sigma-Aldrich, CLS9301). RPFs were precipitated by incubation with 1ul glycogen (Thermo Fisher, R0551) and 3 volumes of 100% ethanol at -20°C overnight. End repair was performed for RPFs using T4 PNK without ATP for 30 minutes at 37°C, followed by 5' phosphorylation with ATP for additional 30 minutes at 37°C. RPFs recovered from the reactions were dissolved in 3ul RNase-free ddH2O and subjected to small RNA library construction using NEBNext Multiplex Small RNA Library Prep Set for Illumina (NEB, E7300). ribo-seq