GSM5989796: uhg4-208F-F-1; Drosophila melanogaster; RNA-Seq
Each sample contained 30 whole flies, flash frozen at 3-5 days of age, and was homogenized using a Fisherbrand™ Bead Mill (ThermoFisher Scientific, Waltham MA). RNA was extracted using the Direct-zol RNA Miniprep kit (Zymo Research, Irvine, CA), resuspended in RNase-free water, and kept at -80°C until further use. We prepared libraries according to Universal RNA-Seq with NuQuant + UDI (Tecan Genomics, Inc., Redwood City, CA) manufacturer instructions. Specifically, 100ng of total RNA was converted into cDNA via integrated DNase treatment. Second strand cDNA was fragmented using a Covaris ME220 Focused-ultrasonicator (Covaris, Woburn, MA) to 350bp. Drosophila AnyDeplete probes were used to deplete remaining ribosomal RNA and final libraries were amplified using 17 PCR cycles. We used TapeStation High Sensitivity D1000 Screentape (Agilent Technologies, Inc., Santa Clara, CA) and a Qubit™ 1X dsDNA HS Assay kit (Invitrogen, Carlsbad, CA) to measure the final library insert sizes and concentration, respectively.