GSM2770691: Patient 15837 Polysome-associated RNA (>3 ribosomes); Homo sapiens; RNA-Seq
Frozen lung tissue samples ranging in weight from 25-60 mg were individually ground to a fine powder using a liquid nitrogen cooled ceramic mortar and pestle. For RNA quality control (QC), approximately 20% of each sample was processed with Tri Reagent (Sigma-Aldrich, St. Louis, MO). Once the initial RNA QC was completed, polyribosome preparations were performed.  For this purpose, the remainder of each frozen powdered sample was solubilized in 100 µl of lysis buffer (10 mM Tris-HCl at pH 8.0, 150 mM NaCl, 5 mM MgCl2, 1% Nonidet-P40, 1% sodium deoxycholate, 40 mM dithiothreitol, 500 U/mL RNAsin RNAsin [Promega, Madison, WI], 40 mM VRC [vanadyl ribonucleoside complex], and 150 μg/ml cycloheximide [Sigma-Aldrich, St. Louis, MO]) and mixed by pipetting.  Nuclei and insoluble material were removed by centrifugation (12,000 x g, 10 minutes, at 4°C). Extraction buffer (50 μL; 0.2 M Tris-HCl at pH 7.5, 0.3 M NaCl, 10 mM PMSF) was added to the supernatant and the sample was mixed by pipetting.  The sample was centrifuged (12,000 x g, 5 minutes, at 4°C) and 25% of the supernatant was processed with Tri Reagent to generate a total mRNA sample.  The remaining supernatant was layered onto a 5 mL, linear sucrose gradient (10-50%), 10 mM Tris-HCl pH 7.5, 140 mM NaCl, 3 mM MgCl2, 10 mM DTT (Sigma-Aldrich, St. Louis, MO). Gradients were centrifuged (200,000 x g in a Beckman SW55Ti rotor, Beckman Coulter, Chaska, MN) for 80 min at 4˚C and the gradients were fractionated into ten, 0.5 mL fractions utilizing an ISCO density gradient fractionator with absorbance monitored at 254 nm. Each fraction was collected into a tube containing 50 μL 1% SDS and 20 μL 0.5M EDTA.  The polysome-associated mRNA from fractions 7, 8, 9 and 10 were individually processed with 1 mL of Tri Reagent and 200 μL chloroform and the resultant RNA pellets from these four fractions were combined to create the polysome-associated mRNA sample corresponding to each tissue sample. Smart-Seq2
GEO Accession
GSM2770691