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| Status |
Public on Aug 27, 2021 |
| Title |
RNA-seq_NK_D Stage 4 EOMES |
| Sample type |
SRA |
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| Source name |
Natural Killer cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Natural Killer cells
day of culture: Day 21
donor: Donor D
treatment: retroviral transduction
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| Treatment protocol |
CD34+ HPC underwent retroviral transduction with T-BET or EOMES overexpression vectors or the control vector. After 48 hours, eGFP+CD34+ HPC were sorted by FACS cell sorting.
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| Growth protocol |
HPC were obtained by isolating peripheral blood monocytes form umbilical cord blood by density gradient centrifugation and subsequently CD34+ HPC were enriched by magnetic activated cell sorting (MACS). Isolated cord blood-derived CD34+ HPC were cultured in complete IMDM containing 10% FCS and supplemented with thrombopoietin (TPO) (20 ng/ml), stem cell factor (SCF) (100 ng/ml) (all from Peprotech, London, UK.) and FMS-like tyrosine kinase 3 ligand (FLT3-L) (100 ng/ml, R&D Systems, Minneapolis, MN, U.S.A.) for 48 hours. Sucessfully transduced HPC were sorted and co-cultured with mitomycin-inactivated EL08.1D2 cells in Dulbecco’s modified Eagle medium plus Ham’s F-12 medium (2:1 ratio), supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM glutamine, 10 mM sodium pyruvate (all from Life Technologies), 20% of heat-inactivated human AB serum (Biowest, Nuaillé, France), 24 μM β-mercaptoethanol, 20 μg/mL ascorbic acid and 50 ng/mL sodium selenite (all from Sigma-Aldrich). The following cytokines were added: IL-3 (5 ng/mL, first week only, R&D systems), IL-7 (20 ng/mL), IL-15 (10 ng/mL) (all from Miltenyi Biotec), SCF (20 ng/mL; Peprotech, London, UK.), and Flt3-L (10 ng/mL; R&D Systems, Minneapolis, MN, U.S.A.) . Culture medium was refreshed on day 7 by addition of the same volume of fresh medium with cytokines. At day 14 the non-adherent cells were harvested and transferred to new mitomycin-treated EL08.1D2 feeder cells.
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| Extracted molecule |
polyA 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, Santa Clara, CA, U.S.A), 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 9
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| Data processing |
Fastq files were aligned to human reference genome GRCh38 using STARv2.42 and gencode v25 as guide gtf
Counts were generated on the fly by STAR
QC of fastq files was done with FastQC
Genome_build: GRCh38
Supplementary_files_format_and_content: a tab separated raw count matrix file
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| Submission date |
Feb 09, 2021 |
| Last update date |
Aug 27, 2021 |
| Contact name |
Wouter Van Loocke |
| E-mail(s) |
[email protected]
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| Organization name |
Ghent University
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| Department |
Biomolecular Medicine
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| Street address |
C. Heymanslaan 10
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| City |
Ghent |
| State/province |
East Flanders |
| ZIP/Postal code |
9000 |
| Country |
Belgium |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE166438 |
Transcriptome profiling of human Natural Killer (NK) cells overexpressing the transcription factors T-BET or EOMES |
| GSE166439 |
ATAC-seq and RNA-seq of hematopoietic progenitor cells (HPC) and Natural Killer (NK) cells transduced with transcription factors T-BET or EOMES |
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| Relations |
| BioSample |
SAMN17841216 |
| SRA |
SRX10058287 |
| 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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