Mature_Male_control_replicate3
For transcriptomic analysis fifty males and females were prepared separately for each experimental variant (control and experimental flies at the age of 1, 4 and 6 weeks). Flies were collected, immediately snap frozen in liquid nitrogen and stored at 80 C. Total RNA was extracted from 30 flies (10 flies per replicate) using QIAzol Lysis Reagent (Qiagen, Netherlands) with the isopropanol precipitation. All samples were treated with DNase I (Promega, USA). RNA concentration and quality were evaluated using a Qubit 2.0 fluorometer (Life Technologies, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Library preparation were carried out by previously used protocol {Moskalev, 2018 #73}. Double stranded cDNA library was prepared by using NEBNext Ultra Directional RNA Library Prep kit for Illumina following manufacturers protocol from 1 mg of total RNA. The quantity of libraries was determined using the qPCR method by Rotor-Gene 6000 PCR System (Qiagen, USA) according to the manufacturers protocol. Primers matched sequences within adapters flanking an Illumina sequencing library. Before starting qPCR, a control template was selected to measure the libraries for quantification. The quality of libraries was defined using Agilent 2100 Bioanalyzer (Agilent, USA) according to the manufacturers protocol. cDNA libraries were normalized to 2nM, pooled together in equal volumes, and sequenced with 50 bp single-end reads on the HiSeq2000 platform (Illumina, USA). Illumina HiSeq Analysis Software was used to obtain raw sequencing reads. The sequencing data were stored in FASTQ format. At least 25 million reads were obtained for each pool of flies.