GSM4102146: math-33 rep 1; Caenorhabditis elegans; ncRNA-Seq
10,000 animals per sample were harvested as adults (~68hrs at 20°C and ~48hrs at 25°C) for RNA extraction. Worms were washed off plates using water and then settled on ice to form a pellet. Water was aspirated off and worm pellets were resuspended in 1mL TRIzol reagent (Life Technologies) and freeze-thawed on dry ice followed by vortexing. Worm carcasses were pelleted using centrifugation and the supernatant containing RNA was collected. 0.2 volume chloroform was add to supernatant, vortexed, centrifuged, and then the aqueous phase was transferred to a new tube. Samples were precipitated using isopropanol and rehydrated in 50μL nuclease-free H2O. Small RNA library preparation. Small RNAs (18 to 30-nt) were size selected on denaturing 15% polyacrylamide gels (BioRad 3450091) from total RNA samples. Small RNAs were treated with 5' RNA polyphosphatase (Epicentre RP8092H) and ligated to 3' pre-adenylated adapter with Truncated T4 RNA ligase (NEB M0373L). Small RNAs were then hybridized to the reverse transcription primer, ligated to the 5' adapter with T4 RNA ligase (NEB M0204L), and reverse transcribed with Superscript III (ThermoFisher 18080-051). Small RNA libraries were amplified using Q5 High-Fidelity DNA polymerase (NEB M0491L) and size selected on a 10% polyacrylamide gel (BioRad 3450051). Library concentration was determined using the Qubit 1X dsDNA HS Assay kit and quality was assessed using the Agilent BioAnalyzer. Libraries were sequenced on the Illumina NextSeq500 (SE 75-bp reads) platform.