Total RNA was isolated by homogenization of dechorionated embryos, followed by the RNA extraction protocol with Trizol. The total RNA was then treated with DNase I to eliminate genomic DNA contamination. The enrichment for poly-adenylated RNAs was also performed using Dynabeads oligo (dT)25 purification. Cytosolic RNA was isolated from the supernatant of the embryo extract before loading onto sucrose gradient. The enrichment for poly-adenylated RNAs was also performed using Dynabeads oligo (dT)25 purification. To obtain the ribosome material, the dechorionated embryos were immediately incubated with 100 μg/ml cycloheximide in PBS for 5 min on ice, then the embryos were homogenized with a plastic pellet pestle in 100 μl of a mild ribosome lysis buffer (20 mM Tris-HCl, pH 7.4, 140 mM KCl, 5 mM MgCl2, 0.5 mM DTT, 1% Triton X-100, 100 μg/ml cycloheximide, 0.5 unit/ml RNasin) and incubated for 10 min on ice. The nuclei and whole cells were removed by centrifugation at 16,000 x g for 10 min at 4 °C, and the lipid and other membranes were filtered out from the supernatant using a 100 μm sieve mesh. Finally, the cytosolic supernatant was loaded onto 20% to 50% continuous sucrose density gradients with the lysis buffer (20 mM Tris-HCl, pH 7.4, 140 mM KCl, 5 mM MgCl2, 0.5 mM DTT), followed by ultracentrifugation at 35,000 rpm for 2.5 hr in a SW41 rotor at 4 °C (OptimaL-100 XP 2100 Ultracentrifuge, Beckman). Absorbance in each layer of the sucrose density gradient was measured at an optical density of 254 nm using the Piston Gradient Fractionater (BioComp). The RNA components and the amount of ribosomes were determined based on distinct peaks in the ribosome profiling. Ribosome material was collected by pooling both the 80S (monosome) and the polysome peaks identified in the profile. The ribosome-associated RNA was extracted by adding an equal volume of Trizol reagent to the ribosome material, followed by chloroform extraction and ethanol precipitation. The poly-adenylated RNA of ribosome-associated RNA was optimized using the RiboMinusTM Eukaryote Kit for RNA-Seq to delete ribosomal RNAs (rRNAs) and Dynabeads oligo (dT)25 purification to select RNAs with poly-adenylated tails. Illumina® TruSeq Stranded mRNA Sample Preparation
Experimental Factor: total RNA extraction
fraction