title: GSM6282838: Mef2-GAL4 x M1BP RNAi 1; Drosophila melanogaster; RNA-Seq
library_construction_protocol: RNA isolation was performed on hand dissected indirect flight muscles from 30, 2-days old females. The tissue was collected in batches of 15 flies at a time to minimize the time between samples and TRIzol treatment and transferred to Eppendorf tubes, then centrifuged at 2000 g for 5 min at 4 ºC. The flight muscle pellet was resuspended in TRIzol reagent (Thermofisher cat #15596026), shock froze in liquid nitrogen and kept at -80 ºC. RNA was isolated directly from the TRIzol muscle samples using a tube-based purification kit from Zymo Research (Direct-zol RNA Microprep, Zymo Research, #R2060) RNA-Seq libraries were generated from 600 ng of total RNA using TruSeq Stranded mRNA Library Prep Kit and TruSeq RNA Single Indexes kits A and B (Illumina, San Diego, CA), according to manufacturer's instructions. Briefly, following purification with poly-T oligo attached magnetic beads, the mRNA was fragmented using divalent cations at 94oC for 2 minutes. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. Strand specificity was achieved by replacing dTTP with dUTP during second strand cDNA synthesis using DNA Polymerase I and RNase H. Following addition of a single 'A' base and subsequent ligation of the adapter on double stranded cDNA fragments, the products were purified and enriched with PCR (30 sec at 98oC; [10 sec at 98oC, 30 sec at 60oC, 30 sec at 72oC] x 12 cycles; 5 min at 72oC) to create the cDNA library. Surplus PCR primers were further removed by purification using AMPure XP beads (Beckman-Coulter, Villepinte, France) and the final cDNA libraries were checked for quality and quantified using capillary electrophoresis.