Human Adult Sorted Live Cell Erythroblasts transduced with Sigma shRNA Clone TRCN0000162889 targeting SLC25A39 with puromycin selection RNAseq.
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE94963 | GEO | 2017/05/01
SECONDARY ACCESSION(S): PRJNA374961
REPOSITORIES: GEO
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Project description:Erythroblasts cultured from mobilized CD34+ cells from healthy adult human donors were used to generate an erythroblast transcriptome transduced with Sigma shRNA Clone TRCN0000005418 targeting RIOK3 gene. Cellular maturation was maintained including enucleation. On culture day 14 total RNA was isolated (see PMID: 23798711 for details). These RNA-Seq profiles were generated after flow cytometric sorting (live cell gating of culture Day 14 erythroblasts according to forward and side scatter). Preliminary screening data demonstrated a robust increase in fetal hemoglobin that may be due to off-target effects from this shRNA clone. A non-targeting shRNA dataset was used for control. These samples were originally published in GSE94147 and are included in this series as well.
2017-08-26 | GSE103122 | GEO
Project description:Erythroblasts cultured from mobilized CD34+ cells from six healthy adult human donors were used to generate an erythroblast transcriptome. Cellular maturation was maintained including enucleation. On culture day 14 total RNA was isolated (see PMID: 23798711 for details). These RNA-Seq profiles were generated after flow cytometric sorting (live cell gating of culture Day 14 erythroblasts according to forward and side scatter).
2017-02-01 | GSE93814 | GEO
Project description:Erythroblasts cultured from six healthy commercial available cord blood CD34+ cells were used to generate an cord blood erythroblast transcriptome. Cellular maturation was maintained including enucleation. On culture day 14 total RNA was isolated (see PMID: 23798711 for details). These RNA-Seq profiles were generated after flow cytometric sorting (live cell gating of culture Day 14 erythroblasts according to forward and side scatter).
2017-02-01 | GSE93960 | GEO
Project description:Erythroblasts cultured from mobilized CD34+ cells from six healthy adult human donors were used to generate an erythroblast transcriptome transduced with Sigma non-targeting shRNA negative control . Cellular maturation was maintained including enucleation. On culture day 14 total RNA was isolated (see PMID: 23798711 for details). These RNA-Seq profiles were generated after flow cytometric sorting (live cell gating of culture Day 14 erythroblasts according to forward and side scatter). We would like to thank Gerard Bouffard and the National Human Genome Research Institute for their expertise and assistance with RNA-seq.
2017-02-01 | GSE94147 | GEO
Project description:To explore the effects of LIN28Ain human erythroid cell development, lentiviral transduction was used for LIN28A over-expression in erythroblasts cultured from mobilized CD34+ cells from six healthy adult human donors. LIN28A over-expression in cultured adult erythroblasts reduced the expression of let-7 and significantly increased HbF expression. Cellular maturation was maintained including enucleation. LIN28Aexpression in adult erythroblasts increased the expression of gamma-globin, and the HbF content of the cells rose to levels >30% of their hemoglobin. mRNA was isolated on culture day 14 (see PMID: 23798711 for details). These RNA-Seq profiles were generated after flow cytometric sorting (live cell gating of culture Day 14 erythroblasts according to forward and side scatter). [Acknowledgement] We would like to thank Gerard Bouffard and the National Human Genome Research Institute for their expertise and assistance with RNA-seq.
2017-02-01 | GSE93256 | GEO
Project description:We identified a role for E2F-2 in the regulation of erythroblast nuclear condensation and enucleation. To help define the mechanism by which E2F-2 regulates these processes, we performed RNA-sequencing on undifferentiated hematopoietic cells and sorted, orthochromatic erythroblasts obtained from wildtype and E2F-2 knockout animals. In undifferentiated progenitor cells we find a limited number of differentially expressed genes associated with E2F-2-loss, likely due to compensation by other E2F family members. However, in late-stage erythroblasts, loss of E2F-2 results in the down-regulation of over 1200 genes. Our subsequent analyses focused on the role of a particular mitotic kinase, Citron Rho-interacting kinase, which we find is induced in an E2F-2-dependent manner during terminal erythroid maturation and identify as a novel regulator of erythroblast enucleation.
2016-10-06 | GSE87127 | GEO
Project description:Human erythroblasts purified from cord blood were cultured in vitro and FACS-sorted into five highly purified populations representing distinct differentiation stages: proerythroblasts, early basophilic erythroblasts, late basophilic erythroblasts, polychromatophilic erythroblasts, and orthochromatophilic erythroblasts. The methods for culture and sorting experiments are given in Hu et al. 2013. For each RNA-seq library, RNA was isolated from 1x 106 sorted human erythroblasts using RNeasy Plus Mini kits (Qiagen). Libraries were then prepared using Illumina TruSeqTM RNA kits to obtain 50 nt reads. Collaborators at the New Your Blood Center were responsible for erythroblast culture, FACS purification of erythroblast populations, and acquisition of RNA-seq data. Collaborators at U.C. Berkeley and Lawrence Berkeley National Laboratory performed data analysis and experimental validation of alternative splicing in erythroblasts. Results: Differentiating erythroblasts execute a dynamic alternative splicing program that is enriched in genes affecting cell cycle, organelle organization, chromatin function, and RNA processing. Alternative splicing plays a major role in regulating gene expression to ensure synthesis of appropriate proteome at each stage as the cells remodel in preparation for production of mature red cells. Erythroid differentiation stage-specific transcriptome analysis was performed by RNA-seq analysis of highly purified erythroblast populations
2013-12-25 | E-GEOD-53635 | biostudies-arrayexpress
Project description:Human erythroblasts purified from cord blood were cultured in vitro and FACS-sorted into five highly purified populations representing distinct differentiation stages: proerythroblasts, early basophilic erythroblasts, late basophilic erythroblasts, polychromatophilic erythroblasts, and orthochromatophilic erythroblasts. The methods for culture and sorting experiments are given in Hu et al. 2013. For each RNA-seq library, RNA was isolated from 1x 106 sorted human erythroblasts using RNeasy Plus Mini kits (Qiagen). Libraries were then prepared using Illumina TruSeqTM RNA kits to obtain 50 nt reads. Collaborators at the New Your Blood Center were responsible for erythroblast culture, FACS purification of erythroblast populations, and acquisition of RNA-seq data. Collaborators at U.C. Berkeley and Lawrence Berkeley National Laboratory performed data analysis and experimental validation of alternative splicing in erythroblasts. Results: Differentiating erythroblasts execute a dynamic alternative splicing program that is enriched in genes affecting cell cycle, organelle organization, chromatin function, and RNA processing. Alternative splicing plays a major role in regulating gene expression to ensure synthesis of appropriate proteome at each stage as the cells remodel in preparation for production of mature red cells.
2013-12-25 | GSE53635 | GEO
Project description:Tropomodulins (Tmods) cap the pointed ends of actin filaments in erythroid and nonerythoid cell types. Targeted deletion of mouse Tmod3 leads to embryonic lethality at E14.5-E18.5, with anemia due to defects in definitive erythropoiesis in the fetal liver. BFU-E and CFU-E colony numbers are greatly reduced, indicating defects in progenitor populations. Flow-cytometry of fetal liver erythroblasts shows late stage populations are also decreased, including reduced percentages of enucleated cells. AnnexinV staining indicates increased apoptosis of Tmod3-/- erythroblasts, and cell cycle analysis reveals that there are more Ter119hi cells in S-phase in Tmod3-/- embryos. Notably, enucleating Tmod3-/- erythroblasts are still in the process of proliferation, suggesting impaired cell cycle exit during terminal differentiation. Tmod3-/- late erythroblasts often exhibit multi-lobular nuclear morphologies and aberrant F-actin assembly during enucleation. Furthermore, native erythroblastic island formation was impaired in Tmod3-/- fetal livers, with Tmod3 required in both erythroblasts and macrophages. In conclusion, disruption of Tmod3 leads to impaired definitive erythropoiesis, due to reduced progenitors, impaired erythroblastic island formation, and defective erythroblast cell cycle progression and enucleation. Tmod3-mediated actin remodeling may be required for erythroblast-macrophage adhesion, coordination of cell cycle with differentiation, and F-actin assembly and remodeling during erythroblast enucleation. Total RNAs from Tmod3+/+ and Tmod3-/- fetal livers at E14.5 were extracted and prepared for microarray analysis using the MoGene-1_0-st-v1 Affymetrix chip in the Scripps Research Microarray Core Facility. Each experiment was repeated with three independent embryos.
2013-10-31 | E-GEOD-51960 | biostudies-arrayexpress
Project description:Tropomodulins (Tmods) cap the pointed ends of actin filaments in erythroid and nonerythoid cell types. Targeted deletion of mouse Tmod3 leads to embryonic lethality at E14.5-E18.5, with anemia due to defects in definitive erythropoiesis in the fetal liver. BFU-E and CFU-E colony numbers are greatly reduced, indicating defects in progenitor populations. Flow-cytometry of fetal liver erythroblasts shows late stage populations are also decreased, including reduced percentages of enucleated cells. AnnexinV staining indicates increased apoptosis of Tmod3-/- erythroblasts, and cell cycle analysis reveals that there are more Ter119hi cells in S-phase in Tmod3-/- embryos. Notably, enucleating Tmod3-/- erythroblasts are still in the process of proliferation, suggesting impaired cell cycle exit during terminal differentiation. Tmod3-/- late erythroblasts often exhibit multi-lobular nuclear morphologies and aberrant F-actin assembly during enucleation. Furthermore, native erythroblastic island formation was impaired in Tmod3-/- fetal livers, with Tmod3 required in both erythroblasts and macrophages. In conclusion, disruption of Tmod3 leads to impaired definitive erythropoiesis, due to reduced progenitors, impaired erythroblastic island formation, and defective erythroblast cell cycle progression and enucleation. Tmod3-mediated actin remodeling may be required for erythroblast-macrophage adhesion, coordination of cell cycle with differentiation, and F-actin assembly and remodeling during erythroblast enucleation.
2013-10-31 | GSE51960 | GEO