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Low-input bulk RNA sequencing of thymic, mediastinal adipose tissue, and aortic arch CD4+CD8+ (DP) cells

ABSTRACT: Low-input bulk RNA sequencing of thymic, mediastinal adipose tissue, and aortic arch CD4+CD8+ (DP) cells

PROVIDER: PRJNA748740 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR15209551.fastq.gz	Fastqsanger.gz
	SRR15209552.fastq.gz	Fastqsanger.gz
	SRR15209553.fastq.gz	Fastqsanger.gz
	SRR15209554.fastq.gz	Fastqsanger.gz
	SRR15209555.fastq.gz	Fastqsanger.gz

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Low-input bulk RNA sequencing of thymic, mediastinal adipose tissue, and aortic arch CD4+CD8+ (DP) cells

Project description:Purpose: To establish if and how mediastinal and adipose tissue and aortic arch CD4+CD8+TCRb- cells (DPs) would differ from thymic DPs, we sorted DPs from all three regions by flow cytometry and subjected the cells to a low-input deep transcriptional profiling assay. Methods: Thymi, mediastinal adipose tissue, and aortic arches of five female, 8-week-old C57BL/6J mice were collected and pooled. Aortic arches and mediastinal adipose tissue were digested as previously described (PMID: 14597735) and single cell suspensions were generated from all three tissues. Four pools were generated per tissue. 400 viable CD4+CD8b+TCRb- cells were sorted into 8ul low-input lysis buffer containing RNaseH inhibitor, dNTPs, and 0.1% Triton X-100 in replicates. Material of 200 cells was reverse transcribed and amplified. Illumina libraries were prepared and sequenced. Results: A total of 2147 genes were differentially regulated between the different DP populations. Extrathymic DPs had 858 genes upregulated and 1284 genes downregulated compared to thymic DPs. 94 genes were commonly regulated in the two extrathymic DP subsets, whereas 23 genes were uniquely expressed in MAT DPs, 31 different genes were exclusively expressed in aortic arch DPs. Conclusion: Our study is the first to identify and provide deep transcriptional analysis two extra-thymic DP populations in mice.

2021-09-28 | GSE180582 | GEO

Arch

Project description:Arch Raw sequence reads

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METTL3 governs thymocyte development and thymic involution by regulating ferroptosis

Project description:Given its central role in immune aging, identifying regulators of thymic involution is important. While conventional programmed cell death plays a fundamental role in thymocyte development, how cell death pathways contribute to thymic involution remains unclear. In this study, we found that CD4+CD8+ double positive (DP) thymocytes acquired characteristics of senescence in aged mice undergoing thymic involution, while expression of the m6A methyltransferase METTL3 (enriched in DP cells from young mice) decreased with aging. By conditionally deleting METTL3 in T cells, we revealed a critical role for METTL3 in DP cell survival, and in restraining features of aging in DP thymocytes by preventing ferroptosis signaling, through glutathione peroxidase 4 (GPX4). Mechanistically, GPX4 was maintained by METTL3 at the translational level, independent of its methyltransferase activity. Furthermore, we found that pharmacological inhibition of ferroptosis promoted DP cell survival, and attenuate d features of aging in DP thymocytes. These findings uncover a role for METTL3-regulated ferroptosis in thymic involution, and identify strategies to restore thymic function.

2024-08-18 | GSE271324 | GEO

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Glyco-gene expression in DP thymic T cells from control and mutant mice that carry a mutation in the ligand binding domain of Notch1

Project description:Dr. Stanley's laboratory is interested in determining how O-fucose glycans function in Notch receptor signaling and in modulating the interactions of Notch receptors with their ligands. We have generated mice carrying a point mutation in the ligand binding domain of Notch1 that reduces Notch ligand binding to mutant T cells. We wish to identify Notch target genes including glycosylation genes with altered expression in DP thymic T cells from mutant and control mice. DP cells were prepared from the thymus of 3 control and 3 mutant mice at ~8 weeks of age and RNA extracted from each prep will be analysed. RNA preparations from CD4+CD8+ double positive (DP) T cells from F+2323, F+2324, F+2329 (control) and FF2330, FF2332, FF2333 (Notch1 with a point mutation to prevent fucosylation in EGF12) mice of ~8 weeks were sent to the Microarray Core (E). The RNA was amplified, labeled, and hybridized to the GLYCOv3 microarrays.

2011-04-29 | E-GEOD-28936 | biostudies-arrayexpress

METTL3 dictates the development of thymocytes via restraining ferroptosis program during age related thymic involution

Project description:Thymic involution is a crucial factor in immune system aging and results in age-related impairment of T cell responses. The control of programmed cell death plays a fundamental role in thymocytes development. How thymocytes cell death contribute to thymic involution remains elusive. In this study, we found that METTL3 is highly expressed in double positive (DP) cells from young mice but decreased in aged mice. Mice conditionally deficiency for METTL3 in T cells showed thymic involution phenotypes with decreased survival and increased senescent DP cells. Additionally, METTL3 deficiency elicited ferroptosis program rather than other cell death pathways and led to defective DP cell development. Furthermore, GPX4, a molecule that protects cells from ferroptosis, was essential for thymocyte survival and preventing senescence. Mechanistically, GPX4 was maintained by METTL3 at the translation level in METTL3 methyltransferase activity independent manner. Targeting METTL3 and ferroptosis process promoted DP cell survival and reinvigorated aged DP cells. These findings reveal the central role of the METTL3-GPX4-ferroptosis axis in regulating thymocyte homeostasis and identifying targets for impeding age-related thymic involution and diseases.

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Glyco-gene expression in DP thymic T cells from control and mutant mice that carry a mutation in the ligand binding domain of Notch1

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Diplodia sp. Dp 3 strain:Dp 3

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Diplodia sp. Dp 4 strain:Dp 4

Project description:Diplodia sp. Dp 4 gene expression analysis - Dp 4-C transcriptome

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