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Single nuclei transcriptomic profiling of adult Drosophila upon oxidative stress

ABSTRACT: Single nuclei transcriptomic profiling of adult Drosophila upon oxidative stress

PROVIDER: PRJNA1022137 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR26213362_1.fastq.gz	Fastqsanger.gz
	SRR26213362_2.fastq.gz	Fastqsanger.gz
	SRR26213363_1.fastq.gz	Fastqsanger.gz
	SRR26213363_2.fastq.gz	Fastqsanger.gz
	SRR26213364_1.fastq.gz	Fastqsanger.gz

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Single nuclei transcriptomic profiling of adult Drosophila upon oxidative stress

Project description:Environmental factors, infection, or injury, can cause oxidative stress in diverse tissues and loss of tissue homeostasis. Effective stress response cascades, conserved from invertebrates to mammals, ensure reestablishment of homeostasis and tissue repair. Hemocytes, the Drosophila blood-like cells, rapidly respond to oxidative stress by immune activation. However, the precise signals how they sense oxidative stress and integrate these signals to modulate their immune response during oxidative stress are ill-defined. Furthermore, it remains unclear if all hemocytes respond similarly to oxidative stress or if there is a specification in distinct subsets. here, we employes single nuclei RNA-sequencing (snRNA-seq) to elcuidate the induced transcriptomic chnages in hemocytes and other cell types by Paraquat feeding compared to control food. Paraquat induces a pronounced oxidative stress response in Drosophila and we used this mdoel to study the diversification of immune cells upon this immune challenge in the adult fly.

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Project description:We used oligonucleotide microarrays to address the specificities of transcriptional responses of adult Drosophila to different stresses induced by paraquat and H2O2, two oxidative stressors, and by tunicamycin which induces an endoplasmic reticulum (ER) stress. Flies were tested 24 hours after exposure to continuous stresses induced by ingestion of paraquat, H2O2 or tunicamycin at concentrations leading to similar effects on viability. We used concentrations of 1% H2O2, 5mM paraquat and 12uM of tunicamycin which lead to negligeable mortality at 24 hours. A paraquat concentration of 15mM was also used for comparison with previous studies Both specific and common responses to the three stressors were observed and whole genome functional analysis identified several important classes of stress responsive genes. Within some functional classes, we observed large variabilities of transcriptional changes between isozymes, which may reflect unsuspected functional specificities.

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Project description:We report RNA-seq of single nuclei isolated from the adult C57BL/6 male mouse Hippocampus region. Majority of the nuclei were isolated from 12 weeks old mice (4 different animal), with an additional set of nuclei from 3 months and 2 years old animals. In addition a set of GFP labeled nuclei driven by a VGAT promoter . Microdissections of dentate gyrus, CA1 and CA2/3 regions of the Hippocampus were placed into ice-cold RNA-later for fixation and stored at 4âc overnight, then stored in -80âc. Nuclei were isolated by sucrose gradient centrifugation and kept on ice until sorting using Fluorescence Activated Cell Sorting (FACS) into 96 well plates containing RNA lysis buffer. Single nucleus RNA was first purified then derived cDNA libraries were generated following a modified Smart-seq2 protocol. For VGAT nuclei: high titer AAV1/2 of pAAV-EF1a-DIO-EYFP-KASH-WPRE-hGH-polyA was injected into dorsal and/or ventral Hippocampus, animals were sacrificed two weeks after injections, and GFP labeled nuclei were sorted into plates and processed as described above.

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