|
| Status |
Public on Nov 01, 2019 |
| Title |
Bone marrow stromal cells, S5534 |
| Sample type |
SRA |
| |
|
| Source name |
bone marrow stromal cells
|
| Organism |
Homo sapiens |
| Characteristics |
donor: BM289
gender: F
age: 45
diabetes: h
passage: P3
stimulation: REST
cell type: bone marrow-derived stromal cells (BMSC)
|
| Treatment protocol |
A subset of n = 7 donors was treated for 24 h with or without cytokines (TNF-alpha and IFN-gamma, both 10 ng/mL), which werematched to the corresponding unstimulated cells and processed in parallel.
|
| Growth protocol |
The cells were isolated from metaphyseal bone marrow (BM) biopsies from patients undergoing hip replacement at Charité University Hospital. Briefly, the BM mononuclear cell fraction (BM-MNC) in primary BM and the BMSC fraction post Ficoll-density gradient centrifugation (Histopaque 1077; Sigma-Aldrich) were quantified with an automated electrical impedance-based CASYR Cell Counter (Schaerfe System GmbH). The BMSCcontaining interphase was plated in a 300 cm2 tissue culture flask (ThermoFischer) and cultured under standard conditions (37◦C, 5% CO2) in an expansion medium (Dulbecco’s Modified Eagle Medium-Low Glucose [DMEM-LG; Sigma-Aldrich] containing 10% fetal calf serum [FCS; Biochrom AG], 100 U/mL penicillin, and 100μg/mL streptomycin [Biochrom AG], and 2mM L-alanyl-L-glutamine [GlutaMAX; Gibco]). The non-adherent fraction was removed by washing with PBS (Gibco), the medium was changed every 72 h, and the cells were allowed to reach about 80% confluence before passaging. The BMSCs were then expanded for several passages and were characterized with multiple functional and molecular assays, in line with the minimal criteria of the International Society for Cellular Therapy (ISCT) (69), at passage three (P3, early passage) and six (P6, late passage).
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted by using the Qiagen RNeasy Plus Mini Kit (Qiagen), according to the manufacturer’s instructions.
Poly-(A)-selection was performed utilizing the NEBNext Poly(A)mRNA Magnetic Isolation Module (NEB) according to the manufacturers requirements. mRNA libraries were prepared with the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB). All libraries were analyzed with the Agilent DNA 1000 Kit and quantified using the Qubit® dsDNA BR Assay Kit (ThermoFisher). After equimolar pooling all samples were sequenced on an Illumina HiSeq system with High Output chemistry v4 (50 cycles, single-read)
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| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 1500 |
| |
|
| Data processing |
HiSeq Control Software 2.2.58, Basecall: RTA 1.18.64.0
FastQ generation: bcl2fastq 1.8.4, FastQ trimming: Trimmomatic 0.36 (ILLUMINACLIP:TruSeq3-SE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:3:20 MINLEN:36), alignment: tophat 2.1.0 with bowtie2 2.2.5 and samtools 0.1.19 (--no-novel-juncs)
Reads were summarized per gene using the featureCount algorithm implemented in the R package Rsubread (v1.16.1).
Raw counts of protein-coding genes were normalized either using varianceStabilizingTransformation or normTransform function of the DESeq2 package (v1.6.3) (pseudocount=1, log2-transformation).
Genome_build: iGenome Homo_sapiens/Ensembl/GRCh38
Supplementary_files_format_and_content: Raw counts - tab-deliminated file with reads summerized per gene.
Supplementary_files_format_and_content: Variancestablized transformed data (vst) - tab-deliminated file with normalized and variance stabilizing transformed data of protein-coding genes
Supplementary_files_format_and_content: Normalized data (norm) - tab-deliminated file with normalized and log2-transformed data (pseudocount=1) of protein-coding genes
|
| |
|
| Submission date |
Oct 18, 2019 |
| Last update date |
Nov 01, 2019 |
| Contact name |
Karsten Jürchott |
| E-mail(s) |
karsten.juerchott@charite.de
|
| Organization name |
Charité - Universitätsmedizin Berlin
|
| Department |
Berlin-Brandenburg Center for Regenerative Therapies
|
| Street address |
Augustenburger Platz 1
|
| City |
Berlin |
| ZIP/Postal code |
D-13353 |
| Country |
Germany |
| |
|
| Platform ID |
GPL18460 |
| Series (1) |
| GSE139073 |
Multi-Parameter Analysis of Biobanked Human Bone Marrow Stromal Cells Shows Little Influence for Donor Age and Mild Comorbidities on Phenotypic and Functional Properties |
|
| Relations |
| BioSample |
SAMN13054554 |
| SRA |
SRX7018659 |
