title: GSM3728210: RiboSeq rep3; Rattus norvegicus; OTHER
library_construction_protocol: Ricci E.P., … Moore M.J. (Nat. Struct. Mol. Biol., 2014) For neurons cultured on semipermeable membranes, RNA was extracted following the steps recommended by TRIzol® reagent (ThermoFisher #15596018) manual with minor modifications. Briefly, media was removed and TRIzol® was applied to the projections-side, the surface was scraped using a cell-scraper and lysate was gently pippeted and transferred into a clean tube. Lysate was similarly collected from the cell body side of the membrane. The lysate was vortexed briefly and kept at room temperature for 5 minutes. 200 μl of chloroform per 1 ml of TRIzol® was added for phase separation. The tubes were vigorously shaken by hand and centrifuged at 12,000 x g for 15 min at 4°C. The aqueous phase was transferred to fresh tubes and the chloroform wash was repeated two more times. RNA was precipitated by adding 100% isopropanol. Samples were incubated at room temperature for 15 min, and centrifuged at 12,000 x g at 4°C for 15 min. The supernatant was removed without disturbing the precipitated RNA. The RNA precipitate was rinsed two times 75% ethanol and finally dissolved in RNase-free water by incubating for 10 min at 55-60°C. For neurons cultured on plates, cyclohexamide was added to the media to inhibit translation and stall ribosomes. The plates were placed on ice, media was removed and the cells were rinsed with ice-cold PBS (containing cyclohexamide). Cells were lysed using lysis buffer (10 mM Tris-HCl pH 7.5, 5 mM MgCl2, 100 mM KCl, 1% Triton™ X-100, 2 mM DTT, 100 μg/ml cycloheximide, protease inhibitor (Complete, EDTA-free, Roche)) and centrifuged to pellet the nuclei. The supernatant was recovered to prepare ribosome profiling and polyA+ RNA-seq libraries.    To purify ribosome occupied RNA, the lysate was treated with RNase T1 (Fermentas) and RNase A (Ambion) to break down polysomes into monosomes, followed by density gradient ultracentrifugation. A gradient fractionator (Brandel) was used to identify and collect the monosome enriched fraction. RNA was subsequently isolated from the monosome enriched fraction for library preparation. Polyadenylated RNA from the cytoplasmic fraction was also sequenced.    To extract RNA for ribosome profiling and polyA+ RNAseq, SDS and proteinase K was added to the lysate and samples were incubated at 42°C for 45 minutes. One volume of acid phenol/chlorofom (Ambion AM9720, pH 4.5) was added and the samples were vortexed for 30 seconds followed by centrifugation at 12,000 x g for 15 minutes. The supernatant was transferred to a clean microcentrifuge tube and 0.1 volume of sodium acetate (3 M, pH 5.2) and 10 mM final concentration of MgCl2 were added. To precipitate RNA, 1 volume of 100% isopropanol was added to the solution and centrifuged at 12,000 x g for 35 minutes. The precipitated RNA was rinsed with 70% ethanol, air dried, and reconstituted in water.
attributes.tag: GEO Accession
attributes.value: GSM3728210