|
| Status |
Public on Aug 04, 2021 |
| Title |
1322: Primary Human MSCs - #5 donor - preconditioned in hypoxia |
| Sample type |
SRA |
| |
|
| Source name |
Mesenchymal Stromal Cells
|
| Organism |
Homo sapiens |
| Characteristics |
cells: primary MSCs (Mesenchymal Stromal Cells)
tissue: bone marrow-derived
treatment: 6h incubation in 2% O2 (hypoxia)
donor sex: male
donor age: 39
|
| Treatment protocol |
Two methods of hypoxic preconditioning were used. The first, "physical" hypoxia was achieved by culturing cells for 6h in a gas mixture composed of 2% O2 (balanced with N2) and 5% CO2. The second, "pharmacological" hypoxia was obtained by culturing cells for 6h with the PHDs inhibitor, Vadadustat (AKB-6548, Akebia, Cambridge, MA, USA) in a concentration of 40 μM. Vadadustat was prepared as a 5 mM stock solution in DMSO according to the manufacturer's instructions, meaning that no more than 0.8 % (v/v) DMSO was present in the culture medium. After 6 h of incubation, cells were harvested, washed with PBS and frozen at -80°C for further analysis.
|
| Growth protocol |
Routine MSCs cultures were maintained under conditions of 5% CO2, 95% humidified air at 37°C. For the analyses, we used six BM-MSCs populations that were between passage 4 and 6 and that fulfilled acknowledged identification criteria for MSCs.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The incubation lasted for 6 h, after which we removed the medium from the cell cultures, washed cells with PBS and disrupted them by scraping in 350 μL of RLT isolation buffer from Qiagen RNeasy Mini Kit according to the protocol provided by the manufacturer.
cDNA libraries construction was carried out with 1μg of total RNA using TruSeq Stranded mRNA Library Prep Kit ( Illumina, San Diego, CA, USA) according to the manufacturer’s instruction.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 1500 |
| |
|
| Description |
*** Raw data not submitted due to patient privacy concerns (currently under IRB review) ***
|
| Data processing |
Basecalls performed using CASAVA version 1.7
Paired-end sequencing (2x100 bp) performed using the HiSeq1500 platform with an average of 16 - 20 million read pairs generated for each library.
Quality control checks of the sequencing raw data conducted with FastQC, and adapter-trimming with BBDUK2 tool.
Quantification of transcript abundance and gene expression was performed using Salmon software.
Exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Genome_build: GRCh37 (hg19)
Supplementary_files_format_and_content: tab-delimited text files include raw gene counts for each sample.
Supplementary_files_format_and_content: Matrix table with normalized gene counts generated by DESeq2 used for GSEA analysis
|
| |
|
| Submission date |
Jul 19, 2021 |
| Last update date |
Aug 04, 2021 |
| Contact name |
Katarzyna Zielniok |
| Organization name |
Medical University of Warsaw
|
| Department |
Department of Clinical Immunology
|
| Lab |
Laboratory of Cellular and Genetic Therapies
|
| Street address |
Nowogrodzka 59
|
| City |
Warsaw |
| ZIP/Postal code |
02-006 |
| Country |
Poland |
| |
|
| Platform ID |
GPL18460 |
| Series (1) |
| GSE180371 |
Gene expression profile of human mesenchymal stromal cells (MSCs) exposed to hypoxic and pseudohypoxic preconditioning |
|
| Relations |
| BioSample |
SAMN20306344 |
