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CD133hi, Notchhi, DP (double positive) and DN (double negative) in GBML8 and GBML20, both patient-derived glioblastoma tumorsphere cultures

ABSTRACT: CD133hi, Notchhi, DP and DN cells were FACS-isolated from GBML8 and GBML20 cultures and analyzed with RNA-seq, in order to find cell type-specific gene signatures.

ORGANISM(S): Homo sapiens

PROVIDER: GSE99180 | GEO | 2017/05/22

SECONDARY ACCESSION(S): PRJNA387469

REPOSITORIES: GEO

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RNAseq analysis of thymic CD4-CD8- double-negative (DN) invariant NKT cells from CD4+CD8+ double-positive (DP)-specific Rag2-conditional knock-out mice

Project description:We report the identification of DN NKT cells developed from DN stage thymocytes. We analyzed the gene expression profiles of NKT cells that have developed from DN stage thymocytes isolated from DP-specific E8III-Cre transgenic Rag2-floxed mouse strain, and NKT cells developed from DP thymocytes sorted as YFP-reporter positive NKT cells that were isolated from E8III-Cre transgenic Rosa26-loxP-STOP-loxP mouse.

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Comparative transcriptomic analysis of germinal center B cells between DN subcluster and DP subcluster.

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Project description:T-cell differentiation requires Notch1 signaling. Here we show that an enhancer upstream of Notch1 active in double-negative (DN) thymocytes is responsible for raising Notch1 signaling intra-thymically. This enhancer is required to expand multipotent progenitors intra-thymically while delaying early differentiation until lineage restrictions are established. Early thymic progenitors lacking the enhancer show accelerated differentiation through the DN stages and increased frequency of B-, ILC-, and NK-cell differentiation. Transcription regulators for T-cell lineage restriction and commitment are expressed normally, but ILC- and NK-cell gene expression persists after T-lineage commitment and TCR V-DJ recombination, Cd3 expression and -selection are impaired. This Notch1 enhancer is inactive in double-positive (DP) thymocytes. Its aberrant reactivation at this stage in Ikaros mutants is required for leukemogenesis. Thus, the DN-specific Notch1 enhancer harnesses the regulatory architecture of DN and DP thymocytes to achieve carefully orchestrated changes in Notch1 signaling required for early lineage restrictions and normal T-cell differentiation.

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Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare the difference of NGS-derived transcriptome profiling between DN thymocytes from Toxf/f and Toxf/fPdcd1Cre mice. Methods: mRNA profiles of DN thymocytes sorted by FACS from 3-4-week-old Toxf/f and Toxf/fPdcd1Cre mice were generated by deep sequencing, in triplicate, using Illumina Novaseq6000. The sequence reads that passed quality filters were analyzed at the transcript level with qRT–PCR using TaqMan and SYBR Green assays. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (Build Ensembl_release100) and identified 19,045 transcripts in the DN thymocytes of Toxf/f and Toxf/fPdcd1Cre mice with BWA workflow. RNA-seq data showed approximately 1.4% of the transcripts which were differentially expressed between the DN thymocytes from Toxf/f and Toxf/fPdcd1Cre mice, with the parameter of false discovery rate (FDR) < 0.05 and absolute fold change ≥ 2. Conclusions: Our study exhibits the detailed analysis of DN thymocyte transcriptomes with biologic replicates, generated by RNA-seq technology. The optimized data analysis here provides a framework for comparative investigations of expression profiles. The RNA-seq data allow researchers to identify the transcripts affected by TOX and to better understand the role of TOX in the development of thymocytes.

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