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Next Generation Sequencing Facilities Quantitative Analysis of Wild type iNKT cells at stage2 and stage3 transcriptomes

ABSTRACT: Next Generation Sequencing Facilities Quantitative Analysis of Wild type iNKT cells at stage2 and stage3 transcriptomes

PROVIDER: PRJNA420959 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR6342040_1.fastq.gz	Fastqsanger.gz
	SRR6342040_2.fastq.gz	Fastqsanger.gz
	SRR6342041_1.fastq.gz	Fastqsanger.gz
	SRR6342041_2.fastq.gz	Fastqsanger.gz
	SRR6342042_1.fastq.gz	Fastqsanger.gz

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Next Generation Sequencing Facilities Quantitative Analysis of Wild type iNKT cells at stage2 and stage3 transcriptomes

Project description:Purpose:Next Generation Sequencing applying for compare the iNKT cells at stage2 and stage3 distict transcriptome, included pathway-enrichment analysis and genes expression of interests Methods:iNKT cells at stage2 and stage3 were isolated from thymus in 6 week old wild type mice by cell sorting. RNA was extracted, purified and checked for integrity using an Agilent Bioanalyzer 2100. Libraries were generated for sequencing using the SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian. Libraries were sequenced using an Illumina HiSeq X Ten sequencer. Resluts: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to further analyse Conclusions:Our study represents the detailed analysis of iNKT cells at stage2 and stage3 transcriptomes, with biologic replicates

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Project description:CD1d expression by thymocytes is required to select iNKT cells. When CD1d is expressed only on thymocytes (pLck-CD1d tg mice), iNKT cells are hyperresponsive to antigen stimulation suggesting that, in physiological conditions, these cells undergo functional education mediated by additional CD1d-expressing cells. Here, we investigated the mechanisms of this functional education. We find that peripheral iNKT cells from pLck-CD1d tg mice express significantly less SHP-1, a tyrosine phosphatase negatively regulating TCR signaling, than WT cells. iNKT cells from heterozygous SHP-1-mutated motheaten mice, displaying similar SHP-1 reduction as pLck-CD1d tg iNKT cells, are antigen-hyperresponsive. Restoring normal CD1d expression in pLck-CD1d tg mice normalizes SHP-1 expression and responsiveness of iNKT cells. In WT mice, iNKT cells upregulate SHP-1 and decrease responsiveness upon emigration from thymus to periphery. This depends on contacts with CD1d-expressing DCs. iNKT cell functional education is therefore controlled by DCs via tuning SHP-1 expression level in the periphery. Hepatic iNKT cells from wild-type and transgenic mice (expressing hCD1d molecule under the pLck promoter)

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Project description:Deletion of Uhrf1 resulted in stage 1-specific defects during iNKT cell development. To investigate the molecular mechanism, we sorted WT and Uhrf1-KO stage 1 iNKT cells and performed RNA-seq. By comparing gene expression profile, we found metabolic defects in Uhrf1-KO stage 1 iNKT cells. The expression of CD71 (Tfrc), two subunits of CD98 (Slc3a2 and Slc7a5) and Glut3 (Slc2a3) was reduced in stage 1 iNKT cells. Besides, the downstream pathways of AKT-mTOR axis were significantly reduced. Collectively, our results suggest that Uhrf1 is required for iNKT cell development by regulating the Akt-mTOR signaling pathway. We first sorted WT and Uhrf1-KO stage 1 iNKT cells, extracted the mRNA and performed RNA-seq. We then analyzed the differentially expressed genes and performed KEGG pathway analysis. We used RT-PCR to verify the expression of the key nutrient related genes (Tfrc, Slc3a2, Slc7a5 and Slc2a3) and used flow cytometry to test the protein level of metabolic related molecules. Besides, we also analyzed the expression of genes of mTOR downstream pathways to demonstrate that Uhrf1 mediated AKt-mTOR axis regulates iNKT cell development.

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