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Sample GSM5009208

Query DataSets for GSM5009208

Status

Public on May 18, 2021

Title

2_HG24_D6_NegC

Sample type

SRA

Source name

human adipose tissue derived stem cells

Organism

Homo sapiens

Characteristics

sirna: Negative control
tissue: Adipose tissue
time point: day 6 (mid differentiation)

Treatment protocol

Transfection was performed using NEON electroporation system (Invitrogen, Carlsbad, USA). 1600V/20ms pulse and 100μl electroporation tips were used to transfect 106 cells. Final concentration of 20nM siRNA was used (JARID2 siRNA L-009244-00-0010 and non-targeting siRNA D-001810-10-20 from Dharmacon). After electroporation, cells were seeded in a density of 40,000 cells/cm2.

Growth protocol

Human mesenchymal stem cells (hMSCs) were isolated from adipose tissue of a male donor (16 years old, BMI 24 kg/m2). Adipose tissue was collagenased and the SVF isolated. The SVF cells were plated in flasks at 3500 cells/cm2 and cultured for 12-14 hours in proliferation medium consisting of low glucose DMEM (Lonza, Verviers, Belgium) supplemented with 10% FBS (Hyclone, Thermo Scientific, Cramlington, UK), penicillin (50 U/ml; Gibco, Life Technologies), streptomycin (50 mg/ml; Gibco, Life Technologies), Hepes (0.5 M) and 2 mmol/l glutamine (Hyclone). The cells were cultured until ~80% confluence, trypsinised and seeded at 4500 cells/cm2 for propagation or at 30 000 cells/cm2 for differentiation. From passage two, FGF2 (5 ng/ml; Sigma-Aldrich) was added to the proliferation medium. The cells were cultured for three days until confluent. FGF2 was then removed from the medium and the cells were incubated for another 24 h. Differentiation medium was prepared by mixing 1:1 of Ham’s F12 (Gibco) and low glucose DMEM supplemented with penicillin (50 U/ml; Gibco, Life Technologies), streptomycin (50 mg/ml; Gibco, Life Technologies), Hepes (0.5 mol/l) and 2 mmol/l glutamine (Sigma- Aldrich). The 1:1 mix was further supplemented with 860 nmol/l insulin, 10 μg/ml transferrin and 0.2 nmol/l triiodothyronine, rosiglitazone (1 μmol/l), IBMX (100 μmol/l) and dexamethasone (1 μmol/l), all from Sigma-Aldrich. The differentiation medium contained 7.8 mmol/l glucose. Differentiation medium was added on day 0 after washing cells twice with PBS. IBMX and dexamethasone were removed after 3 days, and rosiglitazone after 10 days. Full differentiation was reached at day 13. Media were exchanged every 2-3 days.

Extracted molecule

total RNA

Extraction protocol

Total RNA from hASC was extracted with Nucleospin RNA mini kit from Macherey Nagel (Macherey Nagel, Dueren, Germany)
Illumina TruSeq stranded mRNA sample
The yield and quality of the amplified libraries were analyzed using Qubit (Thermo Fisher) and the Agilent Tapestation (Agilent). The indexed cDNA libraries were normalized, combined and the pools were sequenced on the Illumina Nextseq 550 (Illumina) for a 75-cycle v2 sequencing run generating 75bp single-end reads

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

NextSeq 550

Data processing

Sample demultiplexing was performed using bcl2fastq (v2.20.0).
Sample quality was assessed using FastQC (v0.11.8) and MultiQC (v1.7).
Reads were aligned to a reference built from Ensembl GRCh38 genome sequences using STAR (v2.6.1d).
Counts for each gene were obtained using featureCounts (v1.5.1) using the GRCh38.99 GTF file from Ensembl
Bioconductor package DESEq2 was used for count normalization and sample group comparisons, generating log2 fold changes, Wald test p-values and p-values adjusted for multiple testing (Benjamini-Hochberg method).
Supplementary_files_format_and_content: raw and normlized counts

Submission date

Jan 07, 2021

Last update date

May 18, 2021

Contact name

Jurga Laurencikiene

Organization name

Karolinska Institutet

Street address

Hälsovägen 7

City

Stockholm