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Next Generation Sequencing of T regulatory cells (GFP+, dTomato+) and ex-T regulatory cells (GFP-, dTomato+) harvested from lymph nodes of bone marrow chimeras (50% Foxp3GFP-tdT or Foxp3GFP-Fth/-tdT and 50% C57BL/6 bone marrow into Rag2-/- recipients)

ABSTRACT: Next Generation Sequencing of T regulatory cells (GFP+, dTomato+) and ex-T regulatory cells (GFP-, dTomato+) harvested from lymph nodes of bone marrow chimeras (50% Foxp3GFP-tdT or Foxp3GFP-Fth/-tdT and 50% C57BL/6 bone marrow into Rag2-/- recipients)

PROVIDER: PRJNA938398 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR23610180.fastq.gz	Fastqsanger.gz
	SRR23610181.fastq.gz	Fastqsanger.gz
	SRR23610182.fastq.gz	Fastqsanger.gz
	SRR23610183.fastq.gz	Fastqsanger.gz
	SRR23610184.fastq.gz	Fastqsanger.gz

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Next Generation Sequencing of T regulatory cells (GFP+, dTomato+) and ex-T regulatory cells (GFP-, dTomato+) harvested from lymph nodes of bone marrow chimeras (50% Foxp3GFP-tdT or Foxp3GFP-Fth/-tdT and 50% C57BL/6 bone marrow into Rag2-/- recipients)

Project description:Purpose: To address whether FTH regulates gene expression in TREG cells, irrespective of bystander inflammation. For this, we compared the gene expression profile of TREG and Ex-TREG cells sorted from the lymph nodes of mixed BM chimeras Results: Using an optimized data analysis workflow, we mapped an average 30.14 million sequence reads per sample to the mouse genome (build mm39) and identified 34,321 transcripts in the mRNA of Treg and Ex-Treg cells from mixed bone marrow chimeras. Differential gene expression was performed using the DESeq2 R package (v.1.32) and gene expression was modeled by genotype and condition. Differentially expressed genes were considered for genes with an adjusted p-value<0.05 and an absolute log2 fold change>0. Conclusions: Analysis of RNAseq data showed that Fth deletion in TREG cells (CD45.2+GFP+tdT+) developing from the BM of Foxp3GFP-Fth∆/∆-tdT mice, up-regulated 149 genes and down-regulation of 96 genes, compared to TREG cells from control Foxp3GFP-tdT mice. Ex-TREG cells (CD45.2+GFP-tdT+) up-regulated 90 genes and down-regulated 36 genes. The genes regulated in TREG and ex-TREG cells originating from the BM of Foxp3GFP-Fth∆/∆-tdT vs. Foxp3GFP-tdT mice were associated with the oxidative stress response regulated by NRF2 . This suggests that FTH exerts cell-autonomous control of TREG cell redox homeostasis, irrespective of bystander inflammation.

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Project description:The HSC niche factor SCF is required for HSC maintenance. Using an Scf-GFP knockin mouse, we have identified a perivascular cell type in the bone marrow expressing high level of Scf. To characterize the novel Scf-GFP+ cells from the bone marrow, we performed microarray analysis on these cells. Total RNA were isolated from 3 independent, freshly aliquots of FACS sorted 5,000 SCF-GFP+ cells or whole bone marrow cells isolated from young adult mice. Purified RNA was amplified using the WT-OvationM-bM-^DM-" Pico RNA Amplification system (NuGEN Technologies). Sense strand cDNA was generated using WT-OvationM-bM-^DM-" Exon Module (NuGEN), then fragmented and labeled using the FL-OvationM-bM-^DM-" cDNA Biotin Module V2 (NuGEN). 2.5M-BM-5g of labeled cDNA were hybridized to Affymetrix Mouse Gene ST 1.0 microarrays.

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