GSM7993251: null.rep1.RNAseq; Caenorhabditis elegans; RNA-Seq
Harvested worms were washed with M9 medium, centrifuged, and the worm pellets were flash-frozen in liquid nitrogen. The worm pellets were thawed and lysed by adding 4X volumes of QIAzol (Qiagen, Cat: 79306) and shaking vigorously at room temperature for 15 min. The total RNA was extracted by the addition of 0.85X volume chloroform, centrifugation, and recovery of the aqueous phase, which was then re-extracted with 1 volume phenol:chloroform:isoamyl alcohol (25:24:1, pH = 5.5). Total RNA was then precipitated by add 1 volume of isopropanol and 0.5 µl GlycoBlue (Invitrogen, Cat: AM9516), followed by incubation at -80oC for at least 30 min, and recovery by centrifugation at 25,000 rcf for 10 min at 4 °C. The supernatants were then removed, and the RNA pellets were subsequently washed twice by 70% (v/v) ethanol, dried in air for 5 min, dissolved in water, and stored at -80 °C. Worm samples for RNA-seq were aliquoted from the ribosome profiling harvests before the lysis step and frozen separately. The total RNA was extracted as described above. To enrich for mRNA, rRNA was depleted as described in detail elsewhere (Duan et al., 2020b). rRNA-depleted mRNA samples were then purified by RNA Clean & Concentrator-5 Kit (ZYMO, Cat: R1015) Libraries were constructed by NEBNext Ultra II RNA Library Prep kit (NEB, Cat: E7775, E7335, E7500) and sequenced by Illumina NextSeq 500 system RNAseq (rRNA depletion)