Ribo-seq of Arabidopsis thaliana: control conditions
400 mg grinded frozen leaf material was thawed on ice in 5 ml extraction buffer (200 mM Tris/HCl, pH 8.0, 200 mM KCl, 35 mM MgCl2, 200 mM sucrose, 1% Triton X-100, 2% polyoxyethylen-10-tridecyl-ether, 5 mM dithiothreitol, 100 g/ml chloramphenicol, 50 g/ml cycloheximide). The extract was centrifuged for 5 min at 13200 g, 4 C to remove non soluble materials and nuclei, the supernatant was again centrifuged for 10 minutes at 15000 g, 4 C. After addition of 5 mM CaCl2 and 750 units of Micrococcal nuclease (Thermo Fisher) the extract was incubated for 1 hour at room temperature and then loaded on a 2 ml sucrose cushion (40 mM Tris-acetate, pH 8.0, 100 mM KCl, 15 mM MgCl2, 1 M sucrose, 5 mM dithiothreitol, 100 g/ml chloramphenicol, 50 g/ml cycloheximide). After 3 h centrifugation at 55000 g at 4 C using a Type 70.1 Ti rotor (Beckman) the pellet containing the ribosomes was solubilised in 1% SDS, 10 mM Tris, pH 8.0, and 1 mM EDTA. Small RNA containing footprints were isolated from the ribosome fraction using the PureLink miRNA Isolation Kit (Invitrogen). From the small RNA sample the 16-42 nt fraction was isolated by electrophoresis. This RNA fraction was treated with T4 polynucleotide kinase to dephosphorylate the 3 ends before adaptor ligation.