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| Status |
Public on Oct 31, 2022 |
| Title |
ST3, Control, Day 7, rep 3 |
| Sample type |
SRA |
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| Source name |
umbilical cord blood
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| Organism |
Homo sapiens |
| Characteristics |
tissue: umbilical cord blood
cell type: NK cell progenitor cells
shRNA: control
treatment: Lentiviral transduction
time: Day 7
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| Treatment protocol |
Precultured CD34+ cells were transferred to RetroNectin (2 µg/cm²) coated plates and lentiviral supernatant was added followed by spinoculation at 950 g during 90 min at 32°C in the presence of polybrene (8 µg/mL). 24 h after lentiviral transduction, the medium was refreshed to remove polybrene. eGFP+ hematopoietic stem cells (HSCs), defined as CD34+lineage-(CD3/CD14/CD19/CD56)CD45RA- cells, were sorted to high purity 48 h after transduction using a FACS ARIA II cell sorter. Following FACS sorting, eGFP+ HSCs were plated on the mitomycin C-inactivated murine embryonic liver cell line EL08-1D2. Cells were co-cultured in NK cell coculture medium (NKCC) containing Dulbecco’s Modified Eagle Medium (DMEM) and Ham’s F-12 nutrient mixture (2:1 ratio) (all from Thermo Fisher Scientific), supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), glutamine (2 mM), sodium pyruvate (10 mM), heat-inactivated human AB serum (20%), β-mercaptoethanol (24 µM), ascorbic acid (20 µg/mL), ethanolamine (50 µM) and sodium selenite (50 ng/mL). The cytokines IL-3 (5 ng/mL), IL-7 (20 ng/mL), IL-15 (10 ng/mL), SCF (20 ng/mL) and Ftl3L (10 ng/mL) were added to the culture medium. Cultures were maintained in a humidified atmosphere of 5% CO2 at 37°C. On day 3 HSCs, defined as CD34+lineage-(CD3/CD14/CD19/CD56)CD45RA- cells and on day 7 eGFP+ stage (ST)3 (CD34-CD94-CD117+HLA-DR-NKp44-) cells were sorted.
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| Growth protocol |
CD34+ cells were isolated from human umbilical cord blood. After isolation of mononuclear cells by Lymphoprep density gradient centrifugation, CD34+ cells were purified using Magnetic Activated Cell Sorting (MACS). Followed by 48 h of preculture in Iscove’s Modified Dulbecco’s Medium (IMDM) containing foetal calf serum (FCS) (10%), penicillin (100 U/mL), streptomycin (100 µg/mL) and glutamine (2 mM), supplemented with thrombopoietin (TPO) (20 ng/mL), stem cell factor (SCF) (100 ng/mL) and FMS-like tyrosine kinase 3 ligand (Ftl3L) (100 ng/mL).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total mRNA was extracted using the RNeasy micro kit (Qiagen). The concentration and quality of the total extracted RNA was checked by using the Quant-it ribogreen RNA assay (Life Technologies) and the RNA 6000 nano chip (Agilent Technologies), respectively.
Illumina sequencing library preparation was performed using the QuantSeq 3' mRNA-Seq Library Prep Kits (Lexogen, Vienna, Austria) according to manufacturer's protocol. Libraries were quantified by qPCR.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Sample 28
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| Data processing |
Fastq files were aligned to human reference genome GRCh38 using STARv2.42 and gencode v35 as guide gtf
Counts were generated on the fly by STAR
QC of fastq files was done with FastQC
Assembly: Genome_build: GRCh38
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| Submission date |
Aug 25, 2022 |
| Last update date |
Oct 31, 2022 |
| Contact name |
Georges Leclercq |
| E-mail(s) |
georges.leclercq@ugent.be
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| Organization name |
Ghent University
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| Street address |
C. Heymanslaan
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| City |
Ghent |
| ZIP/Postal code |
9000 |
| Country |
Belgium |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE212024 |
Transcriptome analysis of in vitro hematopoietic stem cells (HSC) and stage 3 (ST3) NK cell progenitors cells expressing a TXNIP-specific shRNA. |
| GSE212025 |
Transcriptome analysis of in vitro hematopoietic stem cells (HSC) and stage 3 (ST3) NK cell progenitors cells |
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| Relations |
| BioSample |
SAMN30496935 |
| SRA |
SRX17213616 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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