GSM6594020: TRiP_M_1; Drosophila melanogaster; RNA-Seq
Each sample contained 30 whole flash-frozen 3-5 day old flies, homogenized using a Fisherbrand™ Bead Mill (ThermoFisher Scientific, Waltham MA). RNA was extracted using the the Qiagen RNeasy Plus Mini Kit (Qiagen, Hilden, Germany), resuspended in RNase-free water, and kept at -80°C until further use. Libraries were synthesized from RNA using the Universal RNA-seq with Nuquant + UDI kit (Tecan Genomics, Inc., CA) according to manufacturer recommendations. 100ng of total RNA was converted into cDNA using the integrated DNase treatment and sheared into 350bp fragments using the Covaris ME220 Focused-ultrasonicator (Covaris, Woburn, MA). We performed Ribosomal RNA depletion and bead selection using Drosophila AnyDeplete probes and RNAClean XP beads (Beckman Coulter, Brea, CA), respectively. Libraries were purified after 17 cycles of PCR amplification. We measured library fragment sizes on the Agilent Tapestation using the Agilent High Sensitivity DNA 1000 kit (Agilent Technologies) and quantified library concentration using the Qubit 1X dsDNA High Sensitivity Assay kit (Thermo Fisher Scientific).