Polya synthetic luciferase mrna

Exponentially growing cells were harvested by centrifugation. Total RNA was extracted using Trizol and reverse transcribed using Superscript III (Invitrogen) for standard real-time PCR (RT–qPCR) analysis. For polysomal fraction RT–qPCR analysis, RNA was extracted from each sucrose fraction by standard Trizol method. Five nanograms of polyA+ synthetic luciferase mRNA (Promega) was spiked into the RNA of each fraction for normalization (following methods established by Thoreen et al.38 (link)). All RNA samples were reverse transcribed using Superscript III. RNA from tissue microarray (TMA) slides containing paraffin sections pre-classified as ABC- and GCB-DLBCL by an independent pathologist26 (link) was isolated using RecoverAll Total Nucleic Acid Isolation Kit for FFPE (Life Technologies) according to the manufacturer’s protocol25 (link). RT-qPCR reactions were carried out using SYBR green (Quanta) detection system on a Bio-Rad CFX Connect equipment. All primer sequences used are tabulated in Supplementary Table 7.

Polysome Profiling was carried out as previously described in ref. Gkogkas et al. (2013) (link) with modifications. Dorsal hippocampi were rapidly dissected at the indicated times for each condition, washed with ice-cold PBS containing 100 μg/ml cycloheximide and flash-frozen in liquid N_2. Using a pestle and mortar, tissue was pulverized on dry ice and the powder was resuspended in a hypotonic lysis buffer (5 mM Tris−HCl (pH 7.5), 2.5 mM MgCl₂, 1.5 mM KCl, 100 μg/ml cycloheximide, 2 mM DTT, 0.5 % Triton X-100, and 0.5 % sodium deoxycholate). Lysate concentration was double balanced for protein: by using a Bradford-assay (BIORAD) and for RNA: by measuring total RNA concentration using a NANODROP2000 spectrophotometer (Thermo Scientific). Lysates were loaded onto 5–50 % sucrose density gradients (20 mM HEPES-KOH (pH 7.6), 100 mM KCl, 5 mM MgCl₂) and centrifuged at 35,000 rpm for 2.5 h at 4 °C. The optical density (OD) at 254 nm was continuously recorded using an ISCO fractionator (Teledyne ISCO; Lincoln, NE) for each polysomal fraction; after extraction 5 ng of polyA + synthetic luciferase mRNA (Promega) was added to each fraction for subsequent balancing. Polysome to monosome ratio was calculated as the area under the A254 absorbance curve, using the function describing the recorded values, processed with the definite integral command in MATLAB.