GSM7336834: Ribosome profiling of young brain replicate 1; Nothobranchius furzeri; OTHER
10~15 frozen brain from fish were pooled and lysed using cryo-mill. Lysed samples were thawed and spun at 21,000g for 15 minutes to clear the lysate. RNA concentration of supernatant was measured and RNAse I was added to 0.4 units per ug of RNA. The sample was incubated for 45 minutes at room temperature and 0.02ul of SUPERaseIn RNAse Inhibitor was added per ug of RNA to stop digestion. Samples were immediately layered on 1M sucrose cushion and spun at 100,000rpm for 1 hour in TLA 100.3 rotor to isolate ribosomes. Resulting ribosome pellet was resuspended and RNA was extracted using TRIzol reagent following manufacturer's instructions. RNA fragments in the range of ~25-35 nucleotides were gel purified. After dephosphorylation, a DNA linker (CTGTAGGCACCATCAAT) was ligated to the 3' end of the RNA footprints. Samples were then depleted of rRNA using RiboCop (Lexogen) with the addition of custom-designed oligos to enhance depletion efficiency. Remaining RNA fragments were reverse transcribed into cDNA, followed by circularization. Libraries were barcoded during PCR amplification and sent for sequencing.