GSM8851600: F1_.5_rep2; Caenorhabditis elegans; RNA-Seq
After 52 hrs for control groups and 0.03uM rotenon exposure, or 74 hours for 0.5uM rotenone-exposed groups worms were harvested for sample collection. Samples containing approximately 1,000 nematodes from each group were rinsed several times with K medium  until most of the bacteria were cleared out, then flash-frozen and maintained at -80°C. Nematode samples were used for RNA extraction using 1 ml TRIzol Reagent (Invitrogen) according to the manufacturer's protocol with the exception that approximately 100 µL of acid-washed sand (Sigma-Aldrich), measured with a graduated 1.7 mL microcentrifuge tube, was added to each sample at the beginning of the protocol to aid homogenization. RNA was eluted in nuclease-free water and stored at -80ºC until further use. A nanodrop spectrophotometer (ThermoFisher) was used to assess the purity of the extracted RNA and the Qubit RNA HS Assay kit (ThermoFisher) was used to determine concentration. All RNA samples used for mRNA-Seq library preparation had a 260/280 absorbance ratio over 1.8. Libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs) starting with 500 ng total RNA per library and 9 cycles of PCR. Libraries were sequenced on Illumina NovaSeq 6000 to obtain 50 bp paired-end reads.