GSM2346475: Ribosome profiling of wild-type ecotype Landsberg erecta treated with cycloheximide; Arabidopsis thaliana; OTHER
Cells were collected by filtration and washed with ice-cold PBS with 100 µg/ml CHX added. Collected cells were subjected to snap freezing in liquid nitrogen and frozen cell pellets (approx. 400 mg) were ground into a fine powder using a liquid nitrogen cooled pestle and mortar. The frozen pellet was re-suspended and thawed in 1.3 ml ice-cold lysis buffer (10 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 100 mM KCl, 1% Triton X-100, 2 mM dithiothreitol (DTT), 100 µg/ml CHX, 1x complete and EDTA-free protease inhibitor cocktail (Roche)), vortexed and left on ice for 10 min with periodical agitation. Lysates were subsequently passed through QIAshredder spin columns (Qiagen) and cell debris eliminated by centrifugation at 16,000 x g for 10 min at 4°C. The recovered supernatant was subjected to RNase I (Thermo Fisher Scientific Inc.) digestion using 1500 U of enzyme (approx. 200 U per mg total protein content). Digestion of polysomes proceeded for 45 min at 25°C with gentle agitation at 400 rpm and the reaction stopped by addition of 600 U SUPERase•In™ RNase Inhibitor (Thermo Fisher Scientific). Ribosome protected fragments (RPFs) with sizes ranging from 26-34 nucleotides were extracted from 3 x 20 µg of RNA. Size-selected and dephosphorylated RNA fragments were depleted from ribosomal RNA derived contaminants using the magnetic Ribo-Zero™ rRNA Removal Kits (Plant Leaf, Epicentre) according to the manufacturer's instructions. rRNA depleted using the Ribo-Zero™ Magnetic Kit (Plant Leaf, Epicentre). Samples were amplified by PCR using compatible primers (xxx). The resulting ribosome-profiling libraries were duplexed (samples treated with the same inhibitor, LTM or CHX, were joined) and the obtained cDNA libraries were sequenced on a HiSeq 2000 instrument (Illumina) to yield 50bp single-end reads.