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Sample GSM6857897

Query DataSets for GSM6857897

Status

Public on May 24, 2024

Title

BMP2 rep 2

Sample type

SRA

Source name

BMP2 rep 2

Organism

Homo sapiens

Characteristics

cell type: bone marrow mesenchymal stromal cells (hBM-MSCs)
treatment: BMP2

Treatment protocol

rhBMP-2 and hBM-MSC carrier implants were prepared as previously described.

Growth protocol

Healthy human donor’s bone marrow mesenchymal stromal cells (hBM-MSCs) we obtained from different commercial providers (Lonza, Cat.# PT-2501, Cambrex, Cat.# PT-2501, Millipore, Cat.# SCR108, Inbiobank, Cat.# hMSC). All donor cells were obtained under informed consent and tested for the absence of endothelial and hematopoietic markers, presence of specific mesenchymal markers and their ability to differentiate to osteogenic, chondrogenic and adipogenic phenotype, as defined by ISCT (the International Society for Cellular Therapy) criteria. Cells were expanded in MSCBM mesenchymal cell basal medium (Stem Cells Tech, Cat.# 5401) supplemented with 10% Fetal bovine Serum (FBS) (Gibco, Cat.# 10500064) and antibiotics (Gibco, Cat.# 15140122). Subcultures were performed when cells arrived at 80% confluence by conventional enzymatic cell detachment with trypsin-EDTA 0,25% (Gibco, Cat.# 25200056), followed by cell counting and seeding at 6000 cells/cm2 for subculture, or 100000 cells/well in 6-well plates (Corning, Cat.# 3516) for differentiation assays.

Extracted molecule

total RNA

Extraction protocol

Briefly, Gelfoam gelatin sponges (Pfizer, Cat.# 0009-0323-01) were sectioned into 48 pieces, washed with ethanol 70% (Scharlab, Cat.# ET0003005P) and rehydrated in sterile PBS (Gibco, Cat.# 14040). hBM-MSCs were diluted in culture media at 1x106 cells/ml and 100microl (1x105 cells) were carefully inoculated in each scaffold using a 1mL syringe (Braun, Cat.# 9161406V) with 25G needle (Braun, Cat.# 9186166). Cell-seeded scaffolds were transferred to polystyrene ultra-low attachment 24 well plates (Corning, Cat.# 3473) and maintained in cell culture conditions for 3-5 hours. Then, culture media was added and scaffolds were cultured for 3 to 7 days. 5 microL of acetic acid (Fluka, Cat.# 27225) 50 mM reconstituted rhBMP-2 (Noricum, Cat.# rhBMP-2) (concentration at 5 microg/microL) were added. Then, 30 microL of 2% CaCl2 reconstituted thrombin from human plasma (Merck, Cat.# T8885) and 30 microL of water reconstituted fibrinogen from human plasma (Merck, Cat.# F3879) were incorporated.
RNA libraries were prepared for sequencing using standard Illumina protocols

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

replicate 2

Data processing

Trimmed for adapter sequences using Trimmomatic-0.36 to trim overrepresented sequences.
Alignment and annotated the raw data to the Homo sapiens NCBI GRCh38 genome using HISAT2.
Assemble the mapped reads into possible transcripts and generate a transcriptome assembly with Cufflinks.
Calculation of counts with HTSeq.
Variance stabilization performed using log2 scaling.
Assembly: GRCh38
Supplementary files format and content: tab-delimited text file include log2 counts values for each sample

Submission date

Dec 19, 2022

Last update date

May 24, 2024

Contact name

Marcos J. Araúzo-Bravo

E-mail(s)

mararabra@yahoo.co.uk

Phone

+34 943 00 6108

Organization name

Max Planck Institute for Molecular Biomedicine