GSM6559833: 11, EtOH, rep2 [F_11_E_2]; Drosophila melanogaster; RNA-Seq
Fat body was harvested by dissection using 25 flies per replicate, using only those flies whose ovaries displayed the proper response to S-methoprene supplementation or temperature effect. Flies with either S-methoprene or at 25C had large, viable ovaries, whereas flies at 11C with ethanol displayed the regressed ovarian morphology characteristic of diapause. Tissue was homogenized in Trizol™ with steel beads and total RNA was extracted using the Direct-zol™ RNA MicroPrep kit from Zymo Research. RNA concentration and purity was assessed via Nanodrop™ and Qubit™ technologies prior to library preparation, which was completed at Novogene facilities. Briefly, mRNA was isolated from total RNA via using magnetic beads coated with polyT oligomers. mRNA was then fragmented and cDNA synthesized, followed by end repair, A-tailing, Illumina adapter ligation, size selection, PCR amplification and purification. The final concentrations and purity of libraries were then assessed via Qubit™ and qPCR. Fragment size distribution was assessed via bioanalyzer.