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ScRNA-seq of HSPC treated with gemcitabine and carbplatin

ABSTRACT: The study used cryopreserved hematopoietic stem and progenitor cells (HSPCs) harvested from one patient with chronic myelogenous leukemia (CML). The cells were thawed and acclimatized for 48 hours in StemMACS HSC Expansion Media XF (Miltenyi Biotec) supplemented with StemMACS HSC Expansion Cocktail (Miltenyi Biotec) at 37°C in a humidified atmosphere containing 5% CO2. 4 samples of 2000000 HSPCs in 2 ml media each were then prepared and treated for 24 hours: carboplatin-high: 150 µg/ml carboplatin carboplatin-low: 18.75 µg/ml carboplatin gemcitabine: 25 ng/ml gemcitabine control: no drug Single-cells were subsequently extracted using the ddSEQ™ Single-Cell Isolator (Bio-Rad) and later sequenced using the SureCell™ Whole Transcriptome Analysis 3' Library Prep Kit (Illumina) on the NextSeq 500 System (Illumina) using the NextSeq 500/550 High Output Kit v2.5 150 Cycles (Illumina) all following the manufacturer's instructions. FASTQ-files were then processed following the ddSeeker (Romagnoli, D., et al., BMC Genomics 2018, doi:10.1186/s12864-018-5249-x) and Drop-seq (Macosko, E.Z., et al., Cell 2015, doi:10.1016/j.cell.2015.05.002) protocols for processing scRNA-seq data to yield the final digital gene expression for each cell.

OTHER RELATED OMICS DATASETS IN: PRJNA277735GSE70151PRJNA287748PRJNA267124PRJNA277734PRJNA267857PRJNA267855GSE63269

PROVIDER: EGAS00001004381 | EGA |

REPOSITORIES: EGA

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b66ad1a3-01d6-47b5-aced-df7046415056 - samples

Project description:4 samples of 2000000 HSPCs in 2 ml media each were then prepared and treated for 24 hours: carboplatin-high: 150 µg/ml carboplatin carboplatin-low: 18.75 µg/ml carboplatin gemcitabine: 25 ng/ml gemcitabine control: no drug Single-cells were subsequently extracted using the ddSEQ™ Single-Cell Isolator (Bio-Rad) and later sequenced using the SureCell™ Whole Transcriptome Analysis 3' Library Prep Kit (Illumina) on the NextSeq 500 System (Illumina) using the NextSeq 500/550 High Output Kit v2.5 150 Cycles (Illumina) all following the manufacturer's instructions. The raw FASTQ-files are available as read 1 and read 2 files from 4 lanes for each sample giving a total of 16 FASTQ-files. The FASTQ-files were also processed following the ddSeeker (Romagnoli, D., et al., BMC Genomics 2018, doi:10.1186/s12864-018-5249-x) and Drop-seq (Macosko, E.Z., et al., Cell 2015, doi:10.1016/j.cell.2015.05.002) protocols for processing scRNA-seq data to yield the final digital gene expression (dge) for each cell of each sample. This has resulted in one dge text file and one dge summary text file per sample. These 8 text-files with dge data are found in the zip-compressed analysis data-file.

| EGAD00001006080 | EGA

MYCT1 CONTROLS ENVIRONMENTAL SENSING IN HUMAN HEMATOPOIETIC STEM CELLS

Project description:Hematopoietic stem cells (HSC) rely on a unique regulatory machinery that facilitates life-long blood production and enables reconstitution of the entire hematopoietic system upon transplantation. However, the biological processes governing human HSC self-renewal and engraftment ability are poorly understood and challenging to recapitulate ex vivo to facilitate robust human HSC expansion. We discovered a novel HSC regulatory protein, MYCT1 (MYCT target 1), that is selectively expressed in endothelial cells (EC) and undifferentiated human HSPCs but becomes drastically downregulated during HSC culture. Lentiviral knockdown of MYCT1 in human foetal liver and cord blood HSPCs revealed a critical role for MYCT1 in governing human HSPC expansion and engraftment ability. Single cell RNAseq of human CB HSPCs after MYCT1 knockdown and overexpression revealed that MYCT1 governs HSC functional competence and modulates cellular properties essential for HSC stemness, such as low mitochondrial metabolic activity. Indeed, restoring the compromised MYCT1 expression in cultured human CB HSPCs improved ex vivo expansion of the most undifferentiated human HSPCs and enhanced their engraftment ability. We found that MYCT1 is localized in the endosomal membrane and interacts with vesicle trafficking regulators and signalling machinery essential for HSC and EC function. Loss of MYCT1 led to excessive endocytosis and hyperactive signalling responses to cytokines, whereas restoring MYCT1 expression in cultured CB HSPCs balanced the abnormal endocytosis associated with prolonged culture and fine-tuned signalling responses. Our work identifies MYCT1-moderated endocytosis and environmental sensing as an essential regulatory mechanism required to preserve human HSC stemness, and pinpoints silencing of MYCT1 as a critical contributor to the dysfunction of cultured human HSCs that needs to be addressed to improve human HSC culture strategies.

2024-04-15 | PXD042257 | Pride

Project description:Sequencing of miRXplore Universal Reference (Miltenyi Biotec) RNA using mDOT-seq

| PRJNA680672 | ENA

Acinetobacter oleivorans DR1

Project description:Acinetobacter oleivorans DR1 : Cell treated Ampicillin 100 µg/ml in nutrient media

| PRJNA251413 | ENA

Acinetobacter oleivorans DR1

Project description:Acinetobacter oleivorans DR1 : Cell treated Kanamycin 4 µg/ml in nutrient media

| PRJNA251412 | ENA

Tannic acid (160 µg/ mL) treatment effect on transcriptome of Pseudomonas fluorescens Pf-5

Project description:Transcriptomic profiling of Pseudomonas fluorescens Pf-5 comparing culture treated with 160 µg/mL tannic acid against non-treated culture grown in Mueller-Hinton media

2011-11-24 | GSE33908 | GEO

Tannic acid (20 µg/ mL) treatment effect on transcriptome of Pseudomonas fluorescens Pf-5

Project description:Transcriptomic profiling of Pseudomonas fluorescens Pf-5 comparing culture treated with 20 µg/mL tannic acid against non-treated culture grown in Mueller-Hinton media

2011-11-24 | GSE33907 | GEO

Human CD8 T cell IL2 deprivation time course

Project description:IL2 deprivation time course for day 8 human CD8 T cells activated for 3 days with TransAct T-cell activation beads. PBMC (Cambridge Bio) were defrosted and CD8+ T-cells isolated using a human CD8+ T-cell isolation kit. CD8+ T-cells were activated at 1 x 106 cells/mL with 1 in 100 TransAct T-cell activation beads (Miltenyi) and 20 ng/mL IL-2 in complete RMPI (RMPI 1640, 10% FBS, penicillin/streptomycin) for three days. Cells were then washed out of activation and then maintained in complete RMPI with 20 ng/mL IL-2 for five days. To remove IL-2 for IL-2 deprivation, cells were washed twice in pre-warmed media, then replated in complete media without IL-2. Cells were deprived of IL-2 for 1hr, 3hrs, 5hrs, 8hrs, 24hrs or 48hrs.

2024-10-10 | PXD056475 | Pride

CD160+ and CD160- NK cells from human livers

Project description:Purified NK cells from human liver tissues were first enriched by MACS using NK Cell Isolation Kit (Miltenyi Biotec, German) and CD160+/- hepatic NK cells were isolated by FACS Aria cell sorter (BD Biosciences, United States) to attain a purity greater than 95%.

2020-12-31 | GSE109197 | GEO

CD49a+ and CD49a- NK cells from human livers

Project description:Purified NK cells from human liver tissues were first enriched by MACS using NK Cell Isolation Kit (Miltenyi Biotec, German) and CD49a+/- hepatic NK cells were isolated by FACS Aria cell sorter (BD Biosciences, United States) to attain a purity greater than 95%.

2020-07-07 | GSE109198 | GEO

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