Appl-GAL4 - UAS-Gclc D.Melanogaster (females) Young
Transcriptomic analysis was performed using control flies and transgenic flies with Gclc overexpression at the age of 1 (young), 4 (matured) and 6 weeks (old). 40 males and 40 females in 3 replicates were prepared separately for each experimental variant. Fly transcriptome is strongly gender-dependent and one should not pool flies with different sex into one group. To reveal age-dependent and Gclc-induced transcriptomic changes we applied multivariate generalized linear models (GLM) analysis. Total RNA was isolated from homogenized samples of imago heads by QIAzol Lysis Reagent (Qiagen, Netherlands) and further isopropanol precipitation. The RNA concentration was performed with Qubit®2.0 Fluorometer (Invitrogen, USA) and using a NanoDrop® ND-1000 spectrophotometer (NanoDrop Technologies Inc., USA). The A260/A280 ratio of the RNA samples was 1.8–2.0. The integrity of the isolated RNA (RNA integrity number, RIN) was determined using the Bioanalyzer Agilent 2100 (Agilent Technologies, USA). An RIN value for all samples was not less than 8.0. All samples were treated with DNase I (Fermentas, Lithuania). Sample preparation and RNA sequencing were carried by previously using protocol with modifications. To prepare samples for the mRNA sequencing libraries, we used the Illumina TruSeq™ RNA Sample Preparation Kit (Low-Throughput protocol). Total RNA from each sample of Drosophila (2.5 μg)  was used to purify the poly-A containing mRNA molecules by poly-T oligo-attached magnetic beads, with two rounds of purification. During the second elution of poly-A RNA, the RNA was also fragmented and primed for cDNA synthesis according to the manufacturer’s protocol. Then the fragmented mRNA samples were subjected to cDNA synthesis using a SuperScript Double-Stranded cDNA Synthesis kit (Invitrogen, USA). The cDNA was converted into double-stranded (ds) cDNA and then Ampure XP beads were used to separate ds cDNA from the second-strand reaction mix. The ds cDNA was blunt-ended through an end-repair reaction. Next, a single ‘A’ nucleotide was added to the 3′ ends of the blunt fragments to prevent them from ligating to one another during the adapter ligation reaction. The cDNA fragments were then ligated to specific RNA Adapter Indexes supplied in the kit. The In-Line Control DNA was added to each enzymatic reaction. To selectively enrich DNA fragments with adapter molecules on both ends and to amplify the amount of DNA in the library, the PCR process was used according to the manufacturer’s protocol (15 cycles). The quantity of libraries was determined using a qPCR method on a Rotor-Gene 6000 PCR System (Qiagen, USA) according to the manufacturer’s protocol (Sequencing Library qPCR Quantification Guide). The 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 determined on an Agilent 2100 Bioanalyzer (Agilent, USA) per the manufacturer’s instructions. The size and purity of the samples were checked. The final product was a band at approximately 260 bp. Indexed cDNA libraries were normalized to 2nM and pooled together in equal volumes. Sequencing was performed using HiSeq™2000 platform (Illumina, USA), 50 bp single reads were generated. Raw sequencing reads were obtained using Illumina HiSeq Analysis Software and stored in FASTQ format. At least 25 billion reads were obtained for each pool of flies.