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Sample GSM1695255

Query DataSets for GSM1695255

Status

Public on Mar 21, 2016

Title

control replicate2

Sample type

SRA

Source name

Human articular cartilage

Organism

Homo sapiens

Characteristics

cell type: chondrocyte
treatment perfusion: no
treatment pressure: no
age: 16 years-old
gender: male

Treatment protocol

A simplified bioreactor (Fig.1) described earlier (Schröder et al., 2015), was used to culture human chondrocytes in high-density monolayer culture. In brief, loading and perfusion were applied through the same piping by pressurizing the medium directly. Parallel chambers were used for different stimulations: Perfusion (P) (n=6), hydrostatic pressure plus perfusion (HPP) (n=6) and control (C) (n=6), where cells were kept in static culture. For P and HPP, perfusion was applied with a medium flow rate of 2 ml/min for 20 h/day for 4 days. The perfusion flow rate of 2 ml /min was chosen according to cell viability tests (data not shown). In HPP, hydrostatic pressure of 0.1 MPa for 2 h (loading), followed by 2 h rest (off-loading), was applied each day. During this 4 h period perfusion was stopped in P simultaneously

Growth protocol

Human articular cartilage (triple arthrodesis, 16 y, male) was obtained. The study was approved by the responsible Ludwig-Maximilians-University medical center ethics committee. Chondrocytes were isolated by pronase (Roche Diagnostics GmbH, Mannheim, Germany) and collagenase (Sigma-Aldrich Co., St. Louis, USA). Isolated cells were cultured in a humidified atmosphere at 37° C, 5% CO2 until passage 2. Culture medium consisted of Dulbecco’s Modified Eagle Medium (DMEM)/F12 (1:1, Biochrom AG, Berlin, Germany), 10% fetal bovine serum (FBS) (Biochrom AG, Berlin, Germany), 1% MEM amino acids (Biochrom AG, Berlin, Germany), 25 µg/ml ascorbic acid (Sigma-Aldrich Co., St. Louis, USA), 50 IU/ml Penicillin/Streptomycin (Biochrom AG, Berlin, Germany) and 0.25 µg/ml Amphotericin B (Biochrom AG, Berlin, Germany). The cells were plated 24h prior to stimulation at a density of 106 cells/cm2 in 6-well plates

Extracted molecule

total RNA

Extraction protocol

After 4 days of culture, total RNA was isolated using QiazolTM Lysis Reagent (Qiagen, Hilden, Germany) according to manufacturer’s introduction
An amount of 100 ng total RNA was first treated with double-strand specific DNAse (Fermentas Inc., Hanover. MD , United States) to remove any traces of genomic DNA. After heat-inactivation of the DNAse, cDNA was synthesized and converted to Illumina-compatible sequencing libraries with the Encore complete RNAseq kit from NuGen (NuGen, San Carlos, USA). Briefly, first strand cDNA was generated by selective priming, second strand cDNA was synthesized using dUTP and the generated double stranded cDNA was fragmented using a Covaris M220 sonicator (50W peak incident power, 20% duty factor, 200 cycles per burst, 160 sec treatment time). Then cDNA was end-repaired, ligated to Illumina-Adapters and the second-strand was selectively removed. After 18 cycles of PCR the final library was quantified

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 1500

Description

Data processing

Tophat v2.0, gtf from iGenomes
HTseq count
DEseq2, rlog transformed
Genome_build: hg19
Supplementary_files_format_and_content: raw count table from HTseq count and rlog transformed expression values from DEseq2

Submission date

May 26, 2015

Last update date

May 15, 2019

Contact name

Stefan Krebs

E-mail(s)

krebs-st@lmb.uni-muenchen.de

Phone

0049-89-2180 76715

Organization name

Ludwig-Maximilian University