Next Generation Sequencing Facilitates Quantitative Analysis of U87MG Cell Transcriptomes
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ABSTRACT: Next-generation sequencing (NGS) has significantly advanced the elucidation of developmental signaling mechanisms that are important for U87MG cells under differente treatment. We report here the application of RNA-sequencing technology for transcriptome profile of U87MG cells treated with CAR neutrophils, nanodrugs, and CAR neutrophils loaded nanodrugs. Six U87MG samples were performed in IIIumina HiSeq2500. The resulting sequence reads were mapped to human genome (hg19) using HISAT, and the RefSeq transcript levels (RPKMs) were quantified using the python script rpkmforgenes.py. Our RNA-seq data analyzed different cellular signal pathways under treatment. This study shows a detailed analysis of U87MG transcriptomes generated by RNA-seq technology, providing insight into the mechanisms underlying CAR neutrophils lysis the tumor cells.
Project description:Next-generation sequencing (NGS) has significantly advanced the elucidation of developmental signaling mechanisms that are important for different cell lineage formation from human pluripotent stem cells (hPSCs). We report here the application of RNA-sequencing technology for transcriptome profile of human primary and hPSC-derived neutrophils, and compare to those of hPSCs. Thirteen neutrophil samples from H9 hESCs were performed in IIIumina HiSeq2500. The resulting sequence reads were mapped to human genome (hg19) using HISAT, and the RefSeq transcript levels (RPKMs) were quantified using the python script rpkmforgenes.py. Our RNA-seq data confirmed the stable expression of key neutrophil markers including ITGAM, FUT4, FCGR3A, CEACAM8 and ITGB2. This study shows a detailed analysis of neutrophil transcriptomes generated by RNA-seq technology, providing insight into the mechanisms underlying the differentiation of hPSCs into neutrophils.
Project description:Next-generation sequencing (NGS) has significantly advanced the elucidation of developmental signaling mechanisms that are important for different cell lineage formation from human pluripotent stem cells (hPSCs). We report here the application of RNA-sequencing technology for transcriptome profile of human primary and hPSC-derived epicardial cell, and compare to those of other cell lineages including hPSCs, mesoderm, cardiomcyotyes. Eight epicaridal cell samples from four different hPSC lines and four different donors were performed in IIIumina HiSeq2500. The resulting sequence reads (about 20 million reads per sample) were mapped to human genome (hg19) using HISAT, and the RefSeq transcript levels (RPKMs) were quantified using the python script rpkmforgenes.py. Our RNA-seq data confirmed the stable expression of key epicardial cell markers including WT1, TBX18, TCF21, ALDH1A2 and ZO1, and the gene set enrichment analysis (GSEA) showed enrichment in extracellular matrix related pathways and keratinocyte (epithelial) differentiation. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to epicardial function. This study represents the first detailed analysis of epicardial transcriptomes generated by RNA-seq technology, providing insight into the mechanisms underlying the differentiation of hPSCs into epicardial cells.
Project description:Next-generation sequencing (NGS) has significantly advanced the elucidation of developmental signaling mechanisms that are important for different cell lineage formation from human pluripotent stem cells (hPSCs). We report here the application of RNA-sequencing technology for transcriptome profile of human primary cardiac microvascular endothelial cells and hPSC-derived endocardial endothelial cells, and compare to those of other cell lineages including hPSCs, mesoderm, cardiomcyotyes as well as mouse cardiac microvascular and endocardial endothelial cells. Six cardiac endothelial cell samples from two different hPSC lines and one donor were performed in IIIumina HiSeq2500. The resulting sequence reads (about 20 million reads per sample) were mapped to human genome (hg19) using HISAT, and the RefSeq transcript levels (RPKMs) were quantified using the python script rpkmforgenes.py. Our RNA-seq data confirmed the stable expression of key endocardial endothelial cell markers including CDH5, vWF, PECAM1, NFATC1 and NPR3, and the gene set enrichment analysis (GSEA) showed enrichment in angiogenesis and vasculature development. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to endocardial function. This study represents the first detailed analysis of endocardial-like endothelial cell transcriptomes generated by RNA-seq technology, providing insight into the mechanisms underlying the differentiation of hPSCs into endocardium.
Project description:Next-generation sequencing (NGS) has significantly advanced the elucidation of developmental signaling mechanisms that are important for different cell lineage formation from human pluripotent stem cells (hPSCs). We report here the application of RNA-sequencing technology for transcriptome profile of hPSC-derived hematopoietic cells, and compare to those of other cell lineages including hPSCs, mesoderm, primary aorta-gonad-mesonephros (AGM) cells and cord blood hematopoietic stem cells (HSCs). IIIumina HiSeq 2X150 bp sequencing was performed on three hematopoietic cell samples from three hPSC lines by GENEWIZ. The resulting sequence reads (about 34 million reads per sample) were mapped to human genome (hg19) using HISAT, and the RefSeq transcript levels (RPKMs) were quantified using the python script rpkmforgenes.py. Our RNA-seq data confirmed the stable expression of key hematopoietic cell markers including CD45, RUNX1, CD34 and HOXA clusters, and the gene set enrichment analysis (GSEA) showed enrichment in “aorta development”, “cell migration”, “hematopoietic stem cell proliferation”, “Notch signaling regulation”, et al. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to hematopoietic function. This study presented a detailed analysis of hematopoietic transcriptomes generated by RNA-seq technology, providing insight into the mechanisms underlying the differentiation of hPSCs into hematopoietic cells.
Project description:Differential transcriptome analysis between control cells (U87MG), TMZ-resistant cells with continuous TMZ treatment (U87MG R50) and TMZ-resistant cells with interrupted treatment (U87MG OFF R50).
Project description:U87MG is a glioblastoma cell line that shows substantial heterogeneity despite long-term passaging. We used microarrays to identify variations in gene expression that are associated with phenotypic differences among subclones derived from U87MG.
Project description:This study investigated the biological function of CD133 by ectopic expression of CD133 in U87MG cell line. Although CD133 is widely used as a cancer stem cell marker, there are a few studies that examined its own biological functions. While a number of loss-of-function studies about CD133 have shown that CD133 have effects on cancer progression, there are few gain-of-function studies about functions of CD133. Thus, we identified the potential function of CD133 by its overexpression in U87MG glioblastoma cell line. Though there were no significant changes in cell growth and sphere forming ability, elevated IL-1β and its downstream chemokines (CCL3, CXCL3, CXCL5) may function as chemoattractants which affect recruitment of Ly6G+ neutrophils surrounding necrotic regions in vivo and migration of neutrophil-like dHL-60 cells. Taken together, this results imply that CD133 can regulate IL-1β signaling, and promotes the environmental change.
Project description:EGFRvA is a novel and widely-expressed EGFR isoform, whose upregulation is positively related to glioma grades. Intriguingly, it is the upregulation of EGFRvA but not EGFR that significantly correlates with a poor prognosis in glioma patients. Cancer cells expressing EGFRvA (relative to EGFR) display a higher invasive capacity and a lower sensitivity to EGFR tyrosine kinase inhibitors (TKIs). To investigate the significant differently expressed genes between U87MG EGFRvA cells and U87MG EGFR cells,microarray experiments were conducted.
Project description:We established tumorspheres from human glioblastoma U87MG cells by single cell-derived tumorsphere formation. Among these tumorspheres, P4E8 clone showed cancer stem cell like properties such as self-renewal capacity, expression of cancer stem cell markers, resistance to anti-cancer agents and in vivo tumorigenicity. To find novel therapeutic target molecules, we performed differential gene expression analysis between U87MG and P4E8 cells.