title: RNA-Seq of D. melanogaster heads, female 1-month, control, replicate 2
design: ***** Drosophila Strain and Experimental Conditions:  D. melanogaster wild type Canton-S line (#64349, Bloomington, USA) was used in the experiments. Newly eclosed flies were collected and reared in food medium containing 1000 ml water, 7 g agar, 8 g yeast, 30 g sugar, 30 g semolina, and 3 ml propionic acid. Males and females were kept separately in a Binder KBF720-ICH climate chamber (Binder) at 25C and 60% humidity, with light/dark alternation (12h/12h). Flies were kept in narrow Drosophila vials (Genesee Scientific) at a density of 30 individuals per vial, 5 vials per experimental and control groups. Flies were transferred to the fresh medium twice a week. Flies were kept on the nutrient medium spread with the past of hydrolyzed yeast without Torin-2. Dead flies were coted daily. Heads were manually separated from the bodies and immediately frozen in liquid nitrogen. Three replications of the experiment for each group were performed. ***** Transcriptome sequencing:  Total RNA was isolated from the heads of 30 flies (10 male and female flies per replication) using the reagent QIAzol Lysis Reagent (Qiagen) according to the manufacturer's protocol. The library was prepared according to the previously used protocol [24]. The double-stranded cDNA library was obtained using the NEBNext  Ultra  Directional RNA Library Prep kit for Illumina in accordance with the manufacturer's protocol of 1 mg of total RNA. The number of libraries was determined by the qPCR method using the Rotor-Gene 6000 PCR system (Qiagen) in accordance with the manufacturer's protocol. The primers matched the sequences in the adapters, flanking the Illumina sequencing library. The insertion length in cDNA libraries was approximately 260-270 bp. The quality of the libraries was determined using a highly sensitive DNA chip on the Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara) in accordance with the manufacturer's protocol. High-performance sequencing was carried out in the mode of single-end reads (76 bp) on the NextSeq500 System (Illumina). Because of the isolation of RNA by Trizol, there were different values of 3'-bias for different samples, which should be taken into account in further analysis. This is characteristic of Trizol.