GSM8535043: N2 wildtype L4/YA, 4.5 mM Phenformin treated, monosomal mRNA fraction, Replicate 3; Caenorhabditis elegans; RNA-Seq
Polysome profiling using C. elegans lysates was performed as previously described with minor modifications. For each sample, 30,000 animals of the indicated stage, genotype, strain, or treatment were harvested from 10 cm NGM or RNAi agar plates in M9 and washed three times via gravity settling with fresh M9 to remove bacteria. Worm slurries were then concentrated and solubilized in worm solubilization buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM MgCl2, 1 mM EGTA, 0.1 mg/ml cycloheximide, 1% Triton X-100, 0.1% sodium deoxycholate,1x cOmplete EDTA-free protease inhibitor, and 125 U/µl SUPERase•In RNAse inhibitor. Worms were then homogenized using a motorized pellet pestle with 120 strokes on ice. Homogenates were solubilized at 4°C on a tube rotator for 1 hour, and then cleared via centrifugation at 14,000 x g at 4°C for 5 minutes. 100 microliters of each lysate was retained as a total RNA control. The remainder of the lysate was then loaded into a 5% (w/v) – 50%(w/v) sucrose gradient prepared in high salt resolving buffer containing 25 mM Tris-HCl (pH 8.0), 0.14 M NaCl, and 10 mM MgCl2 using the Gradient Master (Biocomp Instruments), and centrifuged in a Beckman L8 Ultracentrifuge in a SW41 rotor at 41,000 RPM for 1.5 hours at at 4°C, with maximum acceleration and braking. For OP50-1 phenformin experiments, gradients were then loaded onto an ÄKTA pure purification FPLC connected with an in-line UV fluorescence sensor coupled to a Bio-Rad chromatography fraction collector, and 10 1mL fractions were collected per gradient. For egl-45 and eif-3.B RNAi experiments, gradients were loaded and fractionated using a Piston Gradient Station with an Triax Flow Cell (Biocomp Instruments) coupled to an automatic 96 tube rack fraction collector (Gilson) with continuous absorbance recording at 260 nm. In either use case, fractions corresponding to the monosome (80S peak) or polysomes (corresponded to all peaks following the monosome) were pooled together, mixed with TriZol LS (Invitrogen), and flash frozen in liquid nitrogen. RNA was extracted from each pooled fraction and that total lysate using the Direct-Zol RNA Miniprep Plut Kit (Zymo Research) and concentrated using the RNA Clean and Concentrator Kit (Zymo Research). Verification of rRNA integrity in each fraction was verified using an RNA Tapestation 4000 (Agilent). RNA extracts from the total lysate, monosomal fractions, or polysomal fractions of each sample were then subjected to bulk RNA-sequencing or qRT-PCR analysis, as indicated below. For bulk RNA-sequencing analyses, 1000 worms of the indicated strain, genotype, or treatment per sample and replicate was harvested using M9, washed three times to remove bacteria, and worm pellets were resuspended into 500 microliters of RNAzol RT. Total RNA was subsequently extracted and the genomic DNA was removed using the Direct-Zol RNA Miniprep Plus Kit (Zymo Research) following manufacturer's instructions. Total extracted RNA was evaluated for quality control using a NanoDrop One C Microvolume UV-Vis Spectrophotometer (Thermo Fisher). Samples were sent to Azenta (Genewiz) for additional quality control, library preparation, and mRNA sequencing. Samples were validated for RNA integrity with a RIN score > 7.5 and DV200 > 60 using an RNA Tapestation 4200 (Agilent). Illumina library preparation was performed using polyA selection for mRNA species. Approximately 20 million paired-end 150bp reads were generated per sample, with ≥ 80% of bases passing a Phred quality score ≥ Q30.