Single-cell RNA sequencing of human embryonic stem cell-derived pancreatic cells enriched for early endocrine progenitors using a novel dual NEUROG3-reporter
Ontology highlight
ABSTRACT:
INSTRUMENT(S): NextSeq 500
ORGANISM(S): Homo sapiens
SUBMITTER: Belin Selcen Beydag-Tasöz
PROVIDER: E-MTAB-11208 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Human embryonic stem cells with one allele of the NEUROG3 gene genetically modified into a fusion gene NEUROG3-LINKER-TAGRFPT-P2A-EGFP-NLS were differentiated into the pancreatic lineage until Stage 4 Day 1 of the in vitro differentiation protocol (Rezania et al., 2014, as modified in Petersen et al., 2017). GFP positive and negative cells were FAC-sorted according to their cell cycle state (G0/G1 or G2M), and the global transcriptomes of these four groups were compared using bulk RNA-seq.
2022-12-31 | E-MTAB-11210 | biostudies-arrayexpress
Project description:The aim of the experiment is to identify candidate genes to phenotype epitope-specific autoreactive B cells. These genes could serve as markers to delineate further populations of autoreactive B cells that were shown to be key players in regulating autoimmunity and in explaining B cell selection.
2023-08-31 | E-MTAB-13291 | biostudies-arrayexpress
Project description:Myocyte enhancer factor 2B (MEF2B) is a transcription factor with somatic mutation hotspots at K4, Y69 and D83 in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). The recurrence of these mutations indicates that they may drive lymphoma development. However, inferring the mechanisms by which they may drive lymphoma development was complicated by our limited understanding of MEF2B’s normal functions. To expand our understanding of the cellular activities of wildtype (WT) and mutant MEF2B, I developed and addressed two hypotheses: (1) identifying genes regulated by WT MEF2B will allow identification of cellular phenotypes affected by MEF2B activity and (2) contrasting the DNA binding sites, effects on gene expression and effects on cellular phenotypes of mutant and WT MEF2B will help refine hypotheses about how MEF2B mutations may contribute to lymphoma development. To address these hypotheses, I first identified genome-wide WT MEF2B binding sites and transcriptome-wide gene expression changes mediated by WT MEF2B. Using these data I identified and validated novel MEF2B target genes. I found that target genes of MEF2B included the cancer genes MYC, TGFB1, CARD11, NDRG1, RHOB, BCL2 and JUN. Identification of target genes led to findings that WT MEF2B promotes expression of mesenchymal markers, promotes HEK293A cell migration, and inhibits DLBCL cell chemotaxis. I then investigated how K4E, Y69H and D83V mutations change MEF2B’s activity. I found that K4E, Y69H and D83V mutations decreased MEF2B DNA binding and decreased MEF2B’s capacity to promote gene expression in both HEK293A and DLBCL cells. These mutations also reduced MEF2B’s capacity to alter HEK293A and DLBCL cell movement. From these data, I hypothesize that MEF2B mutations may promote DLBCL and FL development by reducing expression of MEF2B target genes that would otherwise function to help confine germinal centre B-cells to germinal centres. Overall, my research demonstrates how observations from genome-scale data can be used to identify cellular effects of candidate driver mutations. Moreover, my work provides a unique resource for exploring the role of MEF2B in cell biology: I map for the first time the MEF2B ‘regulome’, demonstrating connections between a relatively understudied transcription factor and genes significant to oncogenesis. ChIP-seq was performed using a V5 antibody on cells expressing V5 tagged WT and mutant MEF2B. Two biological replicates were performed on WT, K4E, Y69H and D83V MEF2B-V5 cells.
2015-07-01 | E-GEOD-67450 | biostudies-arrayexpress
Project description:The goal of the RNA-sequencing experiment was to identify differentially expressed genes, ontologies, and pathways based on cell cycle traverse rate (CCTR) in pre-treatment malignant samples from patients with primary operable invasive triple-negative breast cancer (TNBC). TNBC patients belonged to the well-characterized Nottingham Primary Breast Carcinoma series, the database of which was prospectively maintained for another study, although its design accommodates the present retrospective analysis. Most TNBCs exhibited high Nottingham grade and about half of patients exhibited positive lymph nodes. All patients received chemotherapy with cyclophosphamide-methotrexate-5-fluoruracil after surgery (radical mastectomy or breast-conserving surgery). Stained slides used for quantitation of Ki67 index (KI) and mitotic index (MI) were independently scored by two pathologists blinded to clinical annotation, and the scores were averaged. Cell cycle traverse rate (CCTR) was defined as the ratio of the percentage of mitotic cells to the percentage of Ki67-positive cells in full-face formalin-fixed paraffin-embedded sections from primary tumors. To estimate percentages of mitotic cells for each sample, the average mitotic counts for each mitotic score category were first determined, followed by a conversion into percentage after accounting for sample cellularity. Separate slides from formalin-fixed paraffin-embedded malignant samples were used for RNA-sequencing based on the availability of sufficient tissue for analysis. Only tissue from pre-marked tumor areas in these slides were used for RNA-sequencing. RNA was extracted from deparaffinized tissue and first- and second-strand synthesis were performed using commercial kits. Pair-read sequencing was performed using the Illumina HiSeq2500 instrument. The Human Genome Assembly GRCh38.P10 was used as the reference genome for mapping sequence reads. Transcript expression was quantified using Salmon. Differentially expressed genes, ontologies, and pathways were determined based on mean CCTR as well as the optimal CCTR to predict breast cancer-specific survival.
2023-02-20 | E-MTAB-6603 | biostudies-arrayexpress
Project description:The goal of the RNA-sequencing experiment was to identify differentially expressed genes, ontologies, and pathways based on HER3-EGFR score in pre-treatment resection samples from patients with primary operable invasive triple-negative breast cancer (TNBC). TNBC patients belonged to the well-characterized Nottingham Primary Breast Carcinoma series, the database of which was prospectively maintained for another study, although its design accommodates the present retrospective analysis. All patients received chemotherapy with cyclophosphamide-methotrexate- 5-fluoruracil after surgery (radical mastectomy or breast-conserving surgery). Stained slides were used for quantitation of HER3 and EGFR H-scores, which were scored independently by two pathologists blinded to clinical annotation, whose scores were averaged. Separate slides from the same formalin-fixed paraffin-embedded malignant samples were used for RNA-sequencing based on the availability of sufficient tissue for analysis. Only tissue from pre-marked tumor areas in these slides were used for RNA-sequencing. RNA was extracted from deparaffinized tissue and first- and second- strand synthesis were performed using commercial kits. Pair-read sequencing was performed using the Illumina HiSeq2500 instrument. The Human Genome Assembly GRCh38.P10 was used as the reference genome for mapping sequence reads. Transcript expression was quantified using Salmon. Differentially expressed genes, ontologies, and pathways were determined based on median HER3-EGFR.
2021-05-27 | E-MTAB-6729 | biostudies-arrayexpress
Project description:Myocyte enhancer factor 2B (MEF2B) is a transcription factor with somatic mutation hotspots at K4, Y69 and D83 in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). The recurrence of these mutations indicates that they may drive lymphoma development. However, inferring the mechanisms by which they may drive lymphoma development was complicated by our limited understanding of MEF2B’s normal functions. To expand our understanding of the cellular activities of wildtype (WT) and mutant MEF2B, I developed and addressed two hypotheses: (1) identifying genes regulated by WT MEF2B will allow identification of cellular phenotypes affected by MEF2B activity and (2) contrasting the DNA binding sites, effects on gene expression and effects on cellular phenotypes of mutant and WT MEF2B will help refine hypotheses about how MEF2B mutations may contribute to lymphoma development. To address these hypotheses, I first identified genome-wide WT MEF2B binding sites and transcriptome-wide gene expression changes mediated by WT MEF2B. Using these data I identified and validated novel MEF2B target genes. I found that target genes of MEF2B included the cancer genes MYC, TGFB1, CARD11, NDRG1, RHOB, BCL2 and JUN. Identification of target genes led to findings that WT MEF2B promotes expression of mesenchymal markers, promotes HEK293A cell migration, and inhibits DLBCL cell chemotaxis. I then investigated how K4E, Y69H and D83V mutations change MEF2B’s activity. I found that K4E, Y69H and D83V mutations decreased MEF2B DNA binding and decreased MEF2B’s capacity to promote gene expression in both HEK293A and DLBCL cells. These mutations also reduced MEF2B’s capacity to alter HEK293A and DLBCL cell movement. From these data, I hypothesize that MEF2B mutations may promote DLBCL and FL development by reducing expression of MEF2B target genes that would otherwise function to help confine germinal centre B-cells to germinal centres. Overall, my research demonstrates how observations from genome-scale data can be used to identify cellular effects of candidate driver mutations. Moreover, my work provides a unique resource for exploring the role of MEF2B in cell biology: I map for the first time the MEF2B ‘regulome’, demonstrating connections between a relatively understudied transcription factor and genes significant to oncogenesis. ChIP-seq was performed using H3K27ac and H3K4me3 antibodies on cells expressing V5 tagged WT MEF2B and on empty vector control cells. Two biological replicates were performed.
2015-07-01 | E-GEOD-67455 | biostudies-arrayexpress
Project description:Single-cell RNA-sequencing (scRNAseq) is revolutionizing biomedicine, propelled by advances in methodology, ease of use, and cost reduction of library preparation. Over the past decade, there have been remarkable technical improvements in most aspects of single-cell transcriptomics. Yet, there has been little to no progress in advancing RNase inhibition despite that maintained RNA integrity is critical during cell collection, storage, and cDNA library generation. Here, we demonstrate that a synthetic thermostable RNase inhibitor yield single-cell libraries of equal or superior quality compared to ubiquitously used protein-based recombinant RNase inhibitors (RRIs). Importantly, the synthetic RNase inhibitor provide additional unique improvements in reproducibility and throughput, enable new experimental workflows including heat cycles, and can reduce the need for dry-ice transports. In summary, replacing RRIs represents a substantial advancement in the field of single-cell transcriptomics.
2024-07-29 | E-MTAB-13873 | biostudies-arrayexpress
Project description:The Wnt/β-catenin signaling pathway plays crucial roles in nearly all parts of embryonic development and adult stem cell homeostasis. Its aberrant activation has been linked to many diseases such as developmental irregularities and various severe forms of cancer, with colorectal cancer (CRC) as a prime example. While much work has been dedicated to uncovering effective therapeutics to block oncogenic Wnt signaling, such interventions have not proven trivial because of the broad activity of Wnt throughout the adult body and the difficulty in finding suitable molecular targets. We have previously identified the developmental transcription factor TBX3 as a participant of the Wnt-mediated transcriptional regulation. Here we examine the genome-wide binding pattern of TBX3 in the human CRC cells lines HCT116 (25 replicates), DLD1 (2 replicates) and SW620 (2 replicates), by employing CUT&RUN (C&R) with Low-Volume and Urea (LoV-U; Zambanini et al., 2022).
2024-01-01 | E-MTAB-13646 | biostudies-arrayexpress
Project description:SOX6 CUT&RUN on HUDEP1 over expressing SOX6-Flag. The experiment is done using and anti Flag Ab to assist the genome wide binding profile of SOX6 in HUDEP1 (Human Umbilical cord blood-Derived Erythroid Progenitor-1).
2023-04-07 | E-MTAB-12800 | biostudies-arrayexpress
Project description:Hypoglycemia is a clinical hallmark of severe malaria, the often-lethal outcome of Plasmodium falciparum infection. Yet, the underlying mechanisms driving the pathogenesis of malaria-associated hypoglycemia remain poorly understood. Here we report that labile heme, an alarmin generated as a byproduct of hemolysis during the blood stage of Plasmodium spp. infection, plays a central role in the development of malaria-associated hypoglycemia. Labile heme recapitulated the hypometabolic response to Plasmodium (chabaudi chabaudi; Pcc) infection in mice, including the development of anorexia, transcriptional repression of hepatic glucose production (HGP) and reduction of glycemia, energy expenditure (EE) as well as core body temperature. While this hypometabolic response is protective against immune-mediated liver damage and anemia, when sustained over time it can lead to hypoglycemia and compromise EE as well as thermoregulation. I response, asexual stages of Plasmodium spp. activate a transcriptional program that reduces virulence in favor of sexual commitment and presumably malaria transmission. In conclusion, malaria-associated hypoglycemia represents a trade-off of a hypometabolic defense strategy against Plasmodium infection.
2021-12-31 | E-MTAB-10939 | biostudies-arrayexpress