title: GSM8453208: Chronic_2AAPA_Rep1; Drosophila melanogaster; RNA-Seq
library_construction_protocol: For extraction and isolation of RNA, 13-21 male flies were euthanized per treatment condition without the use of CO2 sedation by flash freezing in liquid nitrogen. Fly heads from those euthanized were recovered by vortexing to separate heads from bodies and subsequently passing the heads and bodies through two sieves (with the upper sieve retaining the bodies and the lower sieve retaining the heads). Groups of heads were then homogenized in Lysis Binding Mix (from MagMAX mirVana Total RNA Isolation Kit; Thermo Fisher Scientific, Washington, DC; #A27828) using Navy Eppendorf RNA Lysis Kits (Next Advance, Troy, NY; #NAVYE5-RNA) and a Bullet Blender Storm 24 (Next Advance, Troy, NY; #4116-BBY24M). RNA was then isolated from these homogenates using the MagMAX mirVana Total RNA Isolation Kit per the manufacturer's instructions in conjunction with the KingFisher Apex Purification System with 96 Deep-Well head (Thermo Fisher Scientific, Washington, DC; #5400930). For library construction, the quality of RNA was first assessed by TapeStation 2200 (Agilent Technologies, Santa Clara, CA) and NanoDrop (Thermo Fisher Scientific, Washington, DC). cDNA libraries were generated from 500 ng of total RNA using NEBNext Ultra II Directional Poly-A RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA; #E7760). cDNA library quality and quantity were assessed by TapeStation 2200 and Qubit (Thermo Fisher Scientific, Washington, DC). cDNA libraries were then sequenced on a NextSeq 2000 machine using NextSeq 2000 P3 reagents (Illumina, San Diego, CA).