biostudies-arrayexpressUnknownTranscriptomicsGenomicsProteomicsSharifah AnoarIllumina NovaSeq 6000RNA-seq of total RNADrosophila melanogasterDrosophila melanogasterhttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16995Intronic hexanucleotide repeat expansions in the C9orf72 gene are the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). These intronic hexanucleotide repeat expansions result in the production of toxic dipeptide repeat proteins (DPRs), which contribute to neurodegeneration. Our lab previously created fruit fly models expressing 36(G4C2) repeats and individual DPR proteins. This study investigates the transcriptomic effects of C9orf72-associated DPRs in Drosophila melanogaster. Bulk RNA sequencing (RNA-seq) was performed on adult fly heads to assess gene expression changes across three experimental dimensions: 1. Disease progression: We analysed the effect of C9orf72 expressing flies using the elavGS pan-neuronal driver at three time points (days 3, 6, and 9) representing early, mid, and late stages of disease progression. 2. Dipeptide repeat proteins comparison: We compared the transcriptomic profiles of flies expressing different dipeptide repeat proteins (poly-GA36, poly-GR36, and poly-PR36) using the nSybGS driver. 3. Driver effects on gene expression: We evaluated the differences between the two pan-neuronal drivers, elavGS and nSybGS, on gene expression.biostudies-arrayexpressLibrary Construction - The samples were sent to Novogene (UK) Co., Ltd in Cambridge for sequencing. Triplicate samples were prepared for each condition. The company assessed RNA quality and quantity using the 2100 Bioanalyzer Instrument (Agilent) with the RNA Nano Chip (Agilent). Libraries were made on Illumina sequencing PE150 and sequenced as 150 bp paired-end reads, with the mRNA library preparation made with poly A enrichment, resulting in 20 million reads per sample.Nucleic Acid Extraction - RNA extraction: Fly heads were separated from the bodies by mechanical force, 15 heads per sample were placed in cold 2 mL screw cap tubes (Sarstedt) containing 425-600 μm acid-washed glass beads (Merck, Cat No. G8772). 1 mL of cold TRIzol™ reagent (ThermoFisher, Cat No. 15596026) was added to each tube. The tubes were ribolysed at maximum speed for 15 seconds twice and then incubated for 5 minutes at room temperature. After that, 200 μL of chloroform (Fisher Scientific, Cat No. 15668480) was added to each tube, followed by vortexing for 15 seconds and incubation for 2 minutes at room temperature. Samples were centrifuged at full speed for 15 minutes. After centrifugation, the upper colourless phase was collected and transferred into a new Eppendorf tube. Samples were incubated with 500 μL isopropanol (ThermoFisher, Cat No. P/7500/15) for 15 minutes at room temperature, followed by 15 minutes of centrifugation at 4°C at full speed. The RNA pellet was washed twice with 70% ethanol before resuspending in 30 μL nuclease-free water (Ambion, AM9937). The concentration of total RNA purified for each sample was measured using a Nanodrop 2000c spectrophotometer (Thermo Scientific).Sequencing - Subsequently, the clean reads were mapped to the Drosophila reference genome (flybase_drosophila_melanogaster_dmel_r6_49_CDS) with HISAT2 software (Mortazavi et al., 2008). FPKM (short for the expected number of Fragments Per Kilobase of transcript sequence per million base pairs sequenced) was obtained for each gene to evaluate gene expression levels, as provided by the company.Sample Collection - Heads from adult female flies were snap frozen in liquid nitrogen and kept at -80°C before proceeding with RNA extractionOrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesSharifah AnoarTeresa NiccolifalseBulk RNA-seq of Drosophila melanogaster expressing C9orf72 and the associated dipeptide repeat proteins (poly-GA36, poly-GR36, and poly-PR36)Intronic hexanucleotide repeat expansions in the C9orf72 gene are the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). These intronic hexanucleotide repeat expansions result in the production of toxic dipeptide repeat proteins (DPRs), which contribute to neurodegeneration. Our lab previously created fruit fly models expressing 36(G4C2) repeats and individual DPR proteins. This study investigates the transcriptomic effects of C9orf72-associated DPRs in Drosophila melanogaster. Bulk RNA sequencing (RNA-seq) was performed on adult fly heads to assess gene expression changes across three experimental dimensions: 1. Disease progression: We analysed the effect of C9orf72 expressing flies using the elavGS pan-neuronal driver at three time points (days 3, 6, and 9) representing early, mid, and late stages of disease progression. 2. Dipeptide repeat proteins comparison: We compared the transcriptomic profiles of flies expressing different dipeptide repeat proteins (poly-GA36, poly-GR36, and poly-PR36) using the nSybGS driver. 3. Driver effects on gene expression: We evaluated the differences between the two pan-neuronal drivers, elavGS and nSybGS, on gene expression.2026-05-24T00:00:00Z2026-05-26T20:15:25.446Z2026-05-05T16:26:28.244ZE-MTAB-16995ERP193041EFO_0002944EFO_0004170EFO_0009653EFO_0005518EFO_0004184