Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Next Generation Sequencing Facilitates Quantitative Analysis of dendritic cell Transcriptomes during LPS response

ABSTRACT: Purpose:The purpose of this study is to detect activated or silenced genes during LPS-induced dendritic cell. Gene expression differences between two samples could be found using transcriptome profiling (RNA-seq) analysis. Methods:Mouse dendritic cells were generated from bone marrow cells in RPMI-1640 medium with recombinant mouse GM-CSF and IL-4, immature DCs were obtained before or after LPS stimulation. Immature DCs were sorted respectively based on marker CD86 and Iab(MHCII) using flowcytrometer. DC mRNA profiles were generated by deep sequencing,using Illumina. Results: We mapped about 10 million sequence reads per sample to the mouse genome, identified hundreds of genes with significant mRNA variation during LPS stimulation. DC mRNA profiles immature BMDCs were generated by deep sequencing

ORGANISM(S): Mus musculus

SUBMITTER: Qian Zhang

PROVIDER: E-GEOD-69256 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Tet2 is required to resolve inflammation by recruiting Hdac2 to specifically repress IL-6.

Zhang Qian Q Zhao Kai K Shen Qicong Q Han Yanmei Y Gu Yan Y Li Xia X Zhao Dezhi D Liu Yiqi Y Wang Chunmei C Zhang Xiang X Su Xiaoping X Liu Juan J Ge Wei W Levine Ross L RL Li Nan N Cao Xuetao X

Nature 20150819 7569

Epigenetic modifiers have fundamental roles in defining unique cellular identity through the establishment and maintenance of lineage-specific chromatin and methylation status. Several DNA modifications such as 5-hydroxymethylcytosine (5hmC) are catalysed by the ten eleven translocation (Tet) methylcytosine dioxygenase family members, and the roles of Tet proteins in regulating chromatin architecture and gene transcription independently of DNA methylation have been gradually uncovered. However, ...[more]

PMID: 26287468

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Project description:Toll-like receptor (TLR)-induced maturation of dendritic cells (DCs) leads to the production of proinflammatory cytokines as well as the upregulation of various molecules involved in T cell activation. These are believed to be the critical events that account for the induction of the adaptive immune response. Here, we have examined the role of micro-RNA-155 (miR-155) in DC function and the induction of immunity. Using a model where the transfer of self-antigen-pulsed, TLR-matured DCs can induce a functional CD8 T cell response and autoimmunity, we find that DCs lacking miR-155 have an impaired ability to break immune tolerance. Importantly, transfer of self- antigen-pulsed DCs overexpressing miR-155 was sufficient to break tolerance in the absence of TLR stimuli. Although these unstimulated DCs induced T cell function in vivo, there was no evidence for the upregulation of costimulatory ligands or cytokine secretion. Further analysis showed that miR-155 influenced the level of the phosphatase SHIP1 in DCs, and that the lack of SHIP1 in DCs was sufficient to break T cell tolerance in vivo, again in the absence of TLR induced DC maturation. Our study demonstrates that the overexpression of miR-155 in DCs is a critical event that is alone sufficient to break self tolerance and promote a CD8-mediated autoimmune response in vivo. This process is independent of the induction of conventional DC maturation markers, indicating that miR-155 regulation of SHIP represents a unique axis that regulates DC function in vivo. Dendritic cell culture: Bone marrow was flushed from tibias and femurs of mice with HBSS. Bone marrow cells were cultured at 2x106 cells/ml in 10 ml non-tissue culture treated dishes in RPMI 1640 containing 10% LPS-free FBS, Penicillin:streptomycin glutamine 2-mercaptoethanol (cRPMI), with 40ng/ml murine GM-CSF (Peprotech). On day 3 of cultures 10ml of fresh media was added also containing 40ng/ml GM-CSF. On days 6 and 8, 10ml of media was removed and centrifuged to collect cells, which were resuspended in 10ml fresh media containing GM-CSF and were added back to dishes. Non-adherent cells were isolated on day 9. DCs were plated in 24-well plates at 2x106 cells/ml in 1ml of cRPMI with or without the class B CpG ODN1826 (ACGT DNA Technologies corporation, Toronto, ON, Canada) at 10?M final concentration overnight. Array analysis of mi-RNA expression: BMDCs from WT mice were either stimulated with CpG or left in media alone overnight. Whole RNA was isolated using mirVana miRNA isolation kit following the manufacturers instructions. RNA samples were labeled and hybridized to Agilent mouse miRNA 8x15K arrays (Agilent). Data was generated from 8 individual samples (4 unstimulated and 4 CpG-treated).

Dataset Information

Next Generation Sequencing Facilitates Quantitative Analysis of dendritic cell Transcriptomes during LPS response

Publications

Tet2 is required to resolve inflammation by recruiting Hdac2 to specifically repress IL-6.

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