Single cell transcriptomic analysis of thymic epithelial cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: CBDM Lab
PROVIDER: E-GEOD-70798 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Normal thymic T cell development is enabled by a stromal microenvironment most importantly composed of distinct epithelial cell populations in cortex and medulla. Their differentiation, growth and function require the expression of the transcription factor Foxn1. Direct targets of Foxn1 have, however, remained largely undefined. Utilizing newly created static and inducible genetic model systems, we now provide a genome wide map of Foxn1 target genes and the sequences bound by this master regulator. Foxn1 controls not only essential steps early in intrathymic lymphoid development including T cell lineage commitment but is also indispensable for later stages in T cell maturation such as the selection of CD4 and CD8 T cells. Thus, Foxn1 function critically choreographs both early and late events in thymic lympho-stromal cross-talk. Foxn1 ChIP-seq and RNA-seq in mouse models of hypofunctional or conditional knock-out of Foxn1 Brief sample descriptions are shown below: Foxn1 ChIP-seq (GSM1945905) - chromatin immunoprecipitated using an antibody against FOXN1-FLAG (wt*); 2 samples Input ChIP-seq (GSM1945906) - input chromatin; 2 samples 42cT (GSM1945907) - RNA-seq on wt*/-::nu/nu cTEC; 3 samples 42mT (GSM1945908) - RNA-seq on wt*/-::nu/nu mTEC; 2 samples 96cT (GSM1945909) - RNA-seq on wt*/wt*::nu/nu cTEC; 3 samples 96mT (GSM1945910) - RNA-seq on wt*/wt*::nu/nu mTEC; 2 samples NcT (GSM1945911) - RNA-seq on Dox-treated TetO- iFoxn1(del7,8) cTEC; 5 samples PcT (GSM1945912) - RNA-seq on Dox-treated TetO+ iFoxn1(del7,8) cTEC; 5 samples C57BL/6 mice (GSM1945913) - ATAC-seq on wild-type cTEC; 1 sample Please see each sample for more detailed information.
2016-08-09 | E-GEOD-75219 | biostudies-arrayexpress
Project description:We compared in vitro mTECs-high from MyD88fl/fl x Foxn1Cre and MyD88fl/fl mice: Thymi were enzymaticaly digested, cells were MACS enriched for CD45- fraction, and FACS sorted using BD Influx sorter. mTECs-high were gated as EpCAM+CD11c-Ly51-MHCII+CD80+. Total RNA was isolated using RNeasy Plus Micro Kit (Qiagen). 5 samples per condition were used.
2020-03-20 | E-MTAB-8024 | biostudies-arrayexpress
Project description:We compared in vitro mTECs-high stimulated with TLR9 ligand CpG ODN (1826) or non stimulated from MyD88 -/- and MyD88 +/+ mice: Thymi were enzymaticaly digested, cells were MACS enriched for CD45- fraction, and FACS sorted using BD Influx sorter. mTECs-high were gated as EpCAM+CD11c-Ly51-MHCII+CD80+. Cell from MyD88 -/- and MyD88 +/+ mice were incubated in RPMI with or without CpG ODN (1826) for 24 hours. Total RNA was isolated using RNeasy Plus Micro Kit (Qiagen). 4 samples per condition were used.
2020-03-20 | E-MTAB-8025 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2014-09-04 | E-GEOD-53111 | biostudies-arrayexpress
Project description:This study set out to assay the (polyA+) transcriptomes of specific FACS sorted populations of mouse thymic epithelial cells (TEC). Two biological replicates of each of seven murine TEC populations were FACS sorted and sequenced.
2014-09-04 | E-GEOD-53110 | biostudies-arrayexpress
Project description:Thymic central tolerance is essential to preventing autoimmunity. In medullary thymic epithelial cells (mTECs), the Autoimmune regulator (Aire) gene plays an essential role in this process by driving the expression of a diverse set of tissue-specific antigens (TSAs), which are presented and help tolerize self-reactive thymocytes. Interestingly, Aire has a highly tissue-restricted pattern of expression, with only mTECs and peripheral extrathymic Aire-expressing cells (eTACs) known to express detectable levels in adults. Despite this high level of tissue specificity, the cis-regulatory elements that control Aire expression have remained obscure. We used sequence conservation analysis and ChIP-seq against the enhancer-associated histone mark H3K27ac to identify a candidate Aire cis-regulatory element. There is enrichment of H3K27ac near this element, ACNS1, in mTECs and the element also has characteristics of being NF-κB-responsive. Finally, we find that this element is essential for Aire expression in vivo and necessary to prevent spontaneous autoimmunity, reflecting the importance of this regulatory DNA element in promoting immune tolerance. Two experimental groups (GFP neg mTECs and GFP pos mTECs), each with three samples, and one control sample (D10 Th2 cells).
2015-10-23 | E-GEOD-74257 | biostudies-arrayexpress
Project description:This study set out to assay the (polyA+) transcriptomes of single mature (MHCII high) mouse medullary thymic epithelial cells (mTEC). Following isolation by FACs, the transcriptomes of single mature mTEC was assayed using the Fluidigm C1 microfluidics platform and Illumina RNA-seq.
2014-09-04 | E-GEOD-60297 | biostudies-arrayexpress
Project description:Purpose: In all vertebrates, the thymus is necessary and sufficient for production of classic adaptive T cells. The key components of the thymus are cortical and medullary thymic epithelial cells (cTECs and mTECs). Despite the capital role of TECs, our understanding of TEC biology is quite rudimentary. For instance, we ignore what might be the extent of divergence in the functional program of these two TECs populations. It also remains unclear why the number of TECs decreases rapidly with age, thereby leading to progressive thymic insufficiency. Methods: Systems level understanding of cell function begins with gene expression profiling, and the transcriptome is currently the only '-ome' that can be reliably tackled in its entirety in freshly harvested primary cells. In order to gain novel insights into TEC biology, we therefore decided to analyse the whole transcriptome of cTECs, mTECs and skin epithelial cells. We elected to analyse gene expression using RNA-seq rather microarrays because RNA-seq has higher sensitivity and dynamic range coupled to lower technical variations. Results: Our deep sequencing approach provides a unique perspective into the transcriptome of TECs. Consistent with their ability to express ectopic genes, we found that mTECs expressed more genes than other cell populations. Out of a total of 15,069 genes expressed in TECs, 25% were differentially expressed by at least 5-fold in cTECs vs. mTECs. Genes expressed at higher levels in cTECs than mTECs regulate numerous cell functions including cell differentiation, cell movement and microtubule dynamics. Almost all positive regulators of the cell cycle were overexpressed in skin ECs relative to TECs. Conclusions: Our RNA-seq data provide novel insights into the transcriptional landscape of TECs, highlight substantial divergences in the transcriptome of TEC subsets and suggest that cell cycle progression is differentially regulated in TECS and skinECs. We believe that our work will therefore represent a valuable resource and will be of great interest to readers working in biological sciences, particularly in the areas of immunology and systems biology. The mRNA profiles of cTEC, mTEC (from 14 thymi of 7-days old C57BL/6 mice) and skinEC (from the trunk and dorsum of seven newborn mice) were generated by RNA-sequencing using Illumina HiSeq2000.
2013-05-07 | E-GEOD-44945 | biostudies-arrayexpress
Project description:Defense against attaching and effacing (A/E) bacteria requires the sequential generation of IL-23 and IL-22 to induce protective mucosal responses. While the critical source of IL-22 has been identified as CD4+ and Nkp46+ innate lymphoid cells (ILCs), the precise source of IL-23 is unclear. Here, we use genetic techniques to deplete specific classical dendritic cell (cDC) subsets and analyze immunity to the A/E pathogen Citrobacter rodentium. We find that Zbtb46+ cDCs, and specifically Notch2-dependent intestinal CD11b+ cDCs, but not Batf3-dependent CD103+ cDCs, are required for IL-23 production and immunity against C. rodentium. Notch2 controls cDC differentiation at a terminal step mediated by lymphotoxin signaling. Importantly, these results provide the first demonstration of a non-redundant function of CD11b+ cDCs in vivo. Analysis of differentially expressed genes in ESAM+ and ESAM- CD11b+ and DEC205+ splenic classical DC subsets. Splenocytes were harvested from WT C57Bl/6 or WT Cx3cr1-gfp mice and cDC subsets sorted to >95% purity on the FACSAriaII.
2013-06-01 | E-GEOD-45679 | biostudies-arrayexpress
Project description:We used the TriFectaTM (IDT, Integrated DNA Technologies, Coralville, IA, USA) anti-Aire RNAi sequence (GGAUUCUCUUUAAGGACUACAAUCTAGAUUGUAGUCCUUAAAGAGAAUCCUC) in order to silencing Aire mRNA. Confluent cultures of 3.10 mTEC cell line were transfected with 10nM of anti-Aire siRNA using Hiperfect reagent (Qiagen, GmbH, Hilden, Germany) following manufacturerM-^Rs instructions. After transfection, cells were cultured during 24 h in RPMI medium as above mentioned and total RNA was extracted using the mirVana kitM-. (Ambion, Austin, TX, USA), which served as template for cDNA synthesis.<br><br>Gene knockdown was confirmed by quantitative reverse transcription PCR (qRT-PCR) using the primers 5_-GCAACTCTGGCCTCAAAGAG-3_ forward and 5_-GGTCTGAATTCCGTTTCCAA-3_ reverse, which allowed amplification<br><br>of a 120 bp PCR product corresponding to a segment of the Aire cDNA. The cDNA samples were prepared using Superscript II<br><br>reverse transcriptase (Invitrogen Corporation, Carlsbad, CA, USA) enzyme, as recommended. Expression of the above mentioned genes was quantified using a 7500 Real Time PCR System (Applied Biosystems)
2011-08-31 | E-MEXP-3213 | biostudies-arrayexpress