Aire, guardian of immunological tolerance, binds to and activates super-enhancers [ATAC-seq]
ABSTRACT: ATAC-seq profiles of MECs and earskin fibroblasts Overall design: DAPI-CD45-Ly51loMHCIIhi MECs and DAPI-CD45-EpCAM-CD31-Ter-119-Sca-1+ earskin fibroblasts were single sorted and ATAC-seq profiling was perfomed.
Aire is a transcription factor that controls T cell tolerance by inducing the expression of a large repertoire of genes specifically in thymic stromal cells. It interacts with scores of protein partners of diverse functional classes. We found that Aire and some of its partners, notably those implicated in the DNA-damage response, preferentially localized to and activated long chromatin stretches that were overloaded with transcriptional regulators, known as super-enhancers. We also identified to ...[more]
Project description:Microarray profiles of MECs from mice treated with topoisomerase inhibitors Overall design: DAPI-CD45-Ly51loMHCIIhi MECs were single sorted from treated mice. Total RNA was prepared using Trizol method followed by amplification and cDNA preparation. cDNA was hybridized to Affymetrix ST1.0 microarrays.
Project description:ChIP-seq profile of Aire and partners in MECs Overall design: DAPI-CD45-Ly51loMHCIIhi MECs were single sorted and ChIP-seq profiling was perfomed. For ChIP-seq data analysis Illumina output single-end reads were trimmed to 50bp.
Project description:Single cell RNAseq using the 10x gemcode platform on bulk mesenchyme from one adult 6w old mouse. Overall design: Lungs were harvested from a 6-week old adult mouse and digested to generate a single-cell suspension. The cell suspension was stained with antibodies directed against Epcam, CD31 and CD45. DAPI was included as a live/dead stain. Cells that were negative for DAPI, EPCAM, CD31 and CD45, were sorted into collection buffer. The purified cells, referred to as bulk mesenchyme, were manually counted and subsequently loaded onto the 10x Genomics instrument, libraries were prepared according to the manufacture's protocol.
Project description:Chromatin Accessibility Profiles of mTECs from Aire-/- and Brg1-/- mice and their littermate controls. Overall design: DAPIneg, CD45neg, Epcam+, UEA+, Ly51lo, MHCIIhi and MHCIIlo mTECs were sorted from respective mice and ATAC-seq profiling was performed.
Project description:To investigate the influence of Interleukin-22 (IL-22) on colonic intestinal stem cells, we assessed gene expression in these cells during homeostasis and after induction of DNA damage. IL-22 is a lymphocyte-derived cytokine that targets exclusively non-hematopoietic cells. The receptor is expressed on intestinal epithelial cells, including Lgr5+ stem cells. The colonic Lgr5+ epithelial stem cells were highly purified as DAPI-EpCam+CD45-CD24MedLgr5+. DNA damage was induced by whole body irradiation with 8 Gy and cells were isolated 24h after exposure. The following populations were analyzed: Wildtype, unirradiated (Ctrl) Il22-/-, unirradiated (Ctrl) Wildtype, 24h after 8Gy Il22-/-, 24h after 8Gy
Project description:To further development of our gene expression approach to biodosimetry, we have employed microRNA microarray expression profiling to identify genes with the potential to distinguish liver metastasis related microRNA. Colorectal cancer patients were administered anesthesia and 20 mL BM was taken from the right and left anterior iliac crests before surgery. Mononucleated cells were collected using a standard Ficoll-Hypaque gradient technique. To enrich for EpCAM+ cells, CD14+ cells were removed from the whole bone marrow using auto MACSTM pro (Milteny Biotec, Bergisch Gladbach, Germany) with anti-CD14 immunomagnetic beads (clone; TÜK4, Milteny Biotec). Next, CD45+ cells were removed by treatment with anti-CD45 immunomagnetic beads (clone; 5B1; Milteny Biotec). The residual CD14−CD45− cells were then incubated with FcR blocking reagent (Milteny Biotec), followed by incubation with anti-EpCAM immunomagnetic beads (clone; HEA-125, Milteny Biotec), and the CD14−CD45−EpCAM+ cells were taken up. Total RNA of these cells we analyzed the microRNA levels of CD14−CD45−EpCAM+ cells obtained from non-metastasis patients (n = 12) and liver metastasis patients (n = 7). Ten-microRNA consensus signature was identified that distinguished between CD14−CD45−EpCAM+ cells from liver metastasis patients and CD14−CD45−EpCAM+ cells from non-liver metastasis patients. MicroRNA expression of CD14-CD45-EpCAM+ cells in human bone marrow was measured. RNA of these cells we analyzed the microRNA levels of CD14−CD45−EpCAM+ cells obtained from non-metastasis patients (n = 12) and liver metastasis patients (n = 7).
Project description:We developed a protocol for the separation of esophageal carcinoma tissue into leukocytes (using CD45 as a marker), epithelial cells (EpCAM), and fibroblasts (two out of PDGFRA, CD90, anti-fibroblast) by fluorescence-activated cell sorting followed by RNA-sequencing
Project description:We have compared the transcriptional profiles from three epithelial cell types to find common and differentially expressed genes. Epithelial cells from small intestine and kidney from C57BL/6 mice were sorted and their transcriptional profiles compared. Cells were gated as viable, Epcam+ CD45- for kidney, and viable, Epcam+ CD45- MHCIIhigh for small intestine MHCIIhi and viable, Epcam int CD45- MHCII low for small intestine MHCIIlo. Overall design: Three independent replicates were analysed per group. Kidney epithelial cells: kidneys were minced, digested with collagenase, dispase and DNAse. Supernatants were stained with Epcam and CD45 for FACSsort; Fluorogold was used to exclude dead cells. Small intestinal epithelial cells: small intestines were cut open, contents removed and minced. Fragments were washed and incubated with EDTA, and supernatants were stained with Epcam, CD45 and MHCII; Fluorogold was used to exclude dead cells. For small intestine, cells from 3 mice were pooled per replicate. For Kidney, cells from 4 mice were pooled per replicate.
Project description:Beside their role in conventional immune regulation, macrophages are now recognised as essential regulator of local tissue homeostasis depending on the tissue in which they reside. Using phenotyping, we found that LYVE-1+ macrophages are the major resident macrophage population in murine aorta and adipose tissues under steady state. Furthermore, imaging analysis revealed the exclusive association of adipose tissue LYVE-1+ macrophages with smooth muscle positive large blood vessels. Hence, we hypothesize that LYVE-1+ macrophages sustain large vessel functional homeostasis. The present experiment aims to better characterize resident LYVE-1+ vs LYVE-1- macrophages in aorta and adipose tissues. Overall design: LYVE-1+ and LYVE-1- aortic macrophages were FACS sorted as DAPI-CD45+CD64+MerTK+CD11b+F4/80+LYVE-1+ and DAPI-CD45+CD64+MerTK+CD11b+F4/80+LYVE-1- respectively from 30 adult C57/BL6 mice (n =3) their RNA extracted for transcriptome profiling. Similarly, LYVE-1+ and LYVE-1- adipose tissue macrophages were FACS sorted as DAPI-CD45+CD64+MerTK+CD11b+F4/80+LYVE-1+ and DAPI-CD45+CD64+MerTK+CD11b+F4/80+LYVE-1- respectively from 20 adult C57/BL6 epididymal and subcutaneous adipose tissue (n =3) their RNA extracted for transcriptome profiling.