GSM8721535: WM2; Drosophila melanogaster; RNA-Seq
As prepupae were collected, they were homogenized in TRIzol (Invitrogen, ThermoFisher #15596018) and stored at -80°C for later RNA extraction. RNA was isolated from three biological replicates, each containing five white prepupa, by TRIzol extraction followed by isopropanol precipitation. Seven micrograms of total RNA from each replicate were DNaseI-treated and cleaned up using Monarch Total RNA Miniprep Kit (NEB T2010S). One microgram of total RNA was used for RNA-Seq library preparation, using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB E7760) with NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB E7490). Individual libraries were sized and quantified using Tapestation 4200 and High Sensitivity D1000 ScreenTape (Agilent 5067-5584), and Qubit fluorometer with dsDNA HS Assay kit (Thermo Q32854). Equimolar amounts of individual libraries were combined in one pool and sequenced by a commercial provider (Azenta Inc.) using 150 bp paired-end chemistry on NovaSeq X Plus with 5% phiX spike-in.