GSM9149568: VX3, Female Gonad, Biol Rep2; Caenorhabditis remanei; RNA-Seq
We cultured populations of young adult C. remanei (VX0003) and C. latens (VX0088) on NGM-agar plates with Escherichia coli OP50 at 25°C. To collect gonad and somatic tissues, ~400 young adult worms were dissected per sample using a 0.3mm x 30mm needle (BD # 305109). To isolate the tissues, the worms were initially cut behind the pharynx while in sterile water. This process led to the release of the gonads outside the body of the worm. Subsequently, the gonadal arms were separated from the carcass by making a second cut. We pooled together ~400 separated gonads to form one “gonad” tissue sample and the rest of the ~400 carcasses to form one “somatic” tissue sample. Gonad and somatic tissue samples were collected in triplicate separately for males and females of each species. All the samples were stored in Tri-reagent at -80°C for ~48-72 hours after which RNA was isolated using Zymo RNA Isolation kit (#R2051). The isolated RNA was then treated with Turbo DNase (#AM2238) and RiboLock RNase Inhibitor (#EO0381) to degrade any DNA in the samples. To get high quality and high yield of RNA, the samples were further purified using Zymo RNA Clean and Concentrator kit (#R1015) according to manufacturer protocols. Each sample yielded an average >75 million paired-end reads (read length 150bp) generated using NovaSeq S4 flowcell. The Stranded poly(A) mRNA library preparation (NEBNext) and next generation sequencing was performed by The Centre for Applied Genomics, The Hospital for Sick Children, Toronto, Canada.