GSM5732299: JU1088 strain; POS1 treatment; rep 2; Caenorhabditis elegans; RNA-Seq
All samples were collected and processed simultaneously and in triplicate, starting with replicate plates of worms. The following protocol follows (He, 2011). Worms stored at -80°C in TRIzol (Invitrogen #15596026) were thawed and then freeze-thawed 6 times by freezing via submersion in liquid nitrogen, heat at 37°C on a heating block, then vortexing for 10 seconds and placed on ice. 200 uL Trizol was added and samples were incubated for 5 minutes at room temperature. 140 uL chloroform was added and samples were shaken at 1450 rpm for 1 minute, followed by a 2 minute incubation at room temperature. These samples were spun at 12,000g for 15 minutes at 4°C. The top aqueous phase was removed to a new tube and used downstream. One volume of 70% ethanol was added to this aqueous phase and then samples were vortex mixed and quickly spun down. 500 uL of this mixture was transferred to RNeasy columns (Qiagen #74104) and RNA extracted following Qiagen's instructions. RNA was eluted in 50uL RNAse-free water. Libraries were prepared with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #7760) following the manufacturer's instructions. 500ng of fresh RNA was used as input for cDNA generation with this kit with 10 cycles of PCR. Libraries quality checked using an Agilent 2100 Bioanalyzer. BluePippin (Sage Science) was used to size-select fragments size 210-650bp in the pooled library using a 2% agarose gel.