GSM9145035: VavCre Rpl5flox/+ Ter119+ polysome replicate 3; Mus musculus; OTHER
Cell lysates were loaded on top of linear gradients of 10-50% w/v sucrose, 20 mM HEPES-KOH (pH 7.4), 5 mM MgCl2, 100 mM KCl, 2 mM dithiothreitol prepared with a Biocomp Gradient Master 108 in polyallomer tubes (Seton Scientific). The gradients were subjected to ultracentrifugation at 41000 rpm for 2 hr at 4 degC in a Beckman Coulter SW-41Ti rotor. Gradients were fractionated and analyzed for absorbance at 260 nm with a Biocomp Piston Gradient Fractionator. RNA was isolated from the cytoplasm and polysome fractions with Trizol LS (Thermo Fisher Scientific). Magnetic mRNA capture, reverse transcription, adapter ligation, and library amplification were accomplished with the Watchmaker mRNA Library Prep Kit (Watchmaker Genomics). Samples were sequenced on an Illumina NovaSeq X Plus for 100 bp paired-end reads.