Primary human CD34+ cells for modeling ETO2-GLIS2 leukemogenesis - ATACseq
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ABSTRACT: To understand the early consequences of cytokine stimulation, we performed chromatin accessibility assay (ATAC-seq) on primary human CD34+ fetal and cord blood cells exposed or not to IL3 and SCF for 5 days in vitro.
Project description:chromatin accessibility (ATAC-seq) experiment. HeLa cells were primed with IFNγ for 24 hours, followed by IFNγ washout. After 48h, naïve and primed cells were induced by IFNγ for 1h and 3h. Cells were harvested at indicated time points and processed for ATAC-seq.
Project description:Joint profiling of chromatin accessibility and gene expression from the same single cell provides critical information about cell types in a tissue and cell states during a dynamic process. These emerging multi-omics techniques help the investigation of cell-type resolved gene regulatory mechanisms. Here, we developed in situ SHERRY after ATAC-seq (ISSAAC-seq), a highly sensitive and flexible single cell multi-omics method to interrogate chromatin accessibility and gene expression from the same single cell. We demonstrated that ISSAAC-seq is sensitive and provides high quality data with orders of magnitude more features than existing methods. Using the joint profiles from thousands of nuclei from the mouse cerebral cortex, we uncovered major and rare cell types together with their cell-type specific regulatory elements and expression profiles. Finally, we revealed distinct dynamics and relationships of transcription and chromatin accessibility during an oligodendrocyte maturation trajectory.
Project description:To identify conserved TNFα-induced changes in chromatin-accessibility in mammals, we performed ATAC-seq in primary vascular endothelial cells (ECs) isolated from the aortas of human (HAEC), mouse (MAEC) and cow (BAEC), before and after TNFα. We overlay our data with multi-species NF-κB binding data and identify multiple modes of NF-κB-chromatin interactions that are conserved during mammalian TNFα response. Our cross-species approach identifies conserved changes in chromatin-accessibility at NF-κB binding sites that are disease-relevant and essential during mammalian acute inflammation.
Project description:Alternative splicing generates differing RNA isoforms that govern phenotypic complexity of eukaryotes. Its malfunction underlies many diseases, including cancer and cardiovascular diseases. Comparative analysis of RNA isoforms at the genome-wide scale has been difficult. Here, we established an experimental and computational pipeline that accurately quantifies transcript isoforms in their entire length from cDNA sequences with a full-length isoform detection accuracy of 97.6%. We generated a searchable, quantitative human transcriptome annotation with 31,025 known and 5,740 novel transcript isoforms (http://steinmetzlab.embl.de/iBrowser/). By analyzing the isoforms in the presence of RNA Binding Motif Protein 20 (RBM20) mutations associated with aggressive dilated cardiomyopathy (DCM), we identified 121 differentially expressed transcript isoforms in 107 cardiac genes. By establishing an isoform-differential expression test, our approach revealed that 11 of these genes displayed no detectable change in overall RNA expression. However, significant differences in the expression of specific isoforms in these genes was observed. These isoform specific effects demonstrate the need of analyzing RNA isoform expression levels rather than total gene expression levels.
Project description:Rituximab (RTX) is widely used as a first-line therapeutic strategy for patients affected by immune thrombocytopenia (ITP). However, a large proportion of patients relapse after successful treatment. The present NGS assay was done to help find the cause for this relapse on the immune repertoire level. Therefore, we performed antibody repertoire sequencing for three RTX relapse patients with subsequent mutation and clonal analysis, as well as for two patients with ongoing ITP and two healthy donors (HD) with subsequent mutation analysis.
Project description:5'RACE sequencing of B-cell receptors from spleen samples of Atlantic cod. Both IgH and all isotypes of IgL were sequenced with C gene specific primers.
Project description:Multicellular Tumor Spheroids (MCTS) were pre-formed for 3 days with 786-O cell line to better mimic the tumor microenviroenment. These spheroids were treated with either vehicle (DMSO), drugs alone (KU-60019 10 μM; CX-4945 5 μM) or in combination (KU + CX) for 48h before RNA extraction.
Project description:Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer (10.7554/eLife.03848). Specifically, the 2.7 kb Mouse Smarcd3-F6 sequence (chr5: 24113559 -24116342 from mm9 assembly) was sub-cloned from a gateway entry vector into the Zebrafish Enhancer Detection (ZED). Tol2-mediated transgenesis was performed and stable lines were created. The Smarcd3-F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3-F6:EGFPhsc70 embryos were dissociated at 10 hours post fertilization for fluorescent activated cell sorting (FACS). 30,000-50,000 GFP positive and negative cells were collected from FACS for ATAC-seq. ATAC-seq libraries were created with Tn5 transposase and single-end sequenced on the Illumina HiSeq 2500 platform
Project description:HEK293T/17 cells were genetically edited by SMART editing to assess the effectiveness of SMART editing. There are 4 target genes: CXCR4, Lmnb1, Nrl and NRXN3. Cultured cells were first synchronized at the G2/M phase. Then, cells were transfected with Cas9 RNP along with SMART or traditional templates. After culture for two to three days, cells were harvested, and genomic DNA was extracted. The targeted loci were amplified by PCR and sequenced on an Illumina MiSeq.
Project description:To identify the B cell subset(s) involved in T-independent (TI) responses in humans, we vaccinated healthy individuals with Pneumovax, a model TI vaccine composed of 23 capsular polysaccharides (capPS). High-throughput repertoire sequencing of day 7-plasma cells (PC-d7) and of different B cell subpopulations before and after vaccination was done to search for clonal filiations between anti-capPS PC-d7 clones and the isolated B cell subsets. Ig-Rep seq was done for PC-d7 of 6 vaccinee, and for different subsets sampled at day 0, 7, 14, 28 and 56 from 3 vacinees.