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Sample GSM1695260 Query DataSets for GSM1695260
Status Public on Mar 21, 2016
Title hydrostatic pressure plus perfusion replicate1
Sample type SRA
Source name Human articular cartilage
Organism Homo sapiens
Characteristics cell type: chondrocyte
treatment perfusion: yes
treatment pressure: yes
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 HPP1
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
Phone 0049-89-2180 76715
Organization name Ludwig-Maximilian University
Department Gene Center
Lab Lafuga Genomics
Street address Feodor-Lynen-Str. 25
City Munich
ZIP/Postal code 81377
Country Germany
Platform ID GPL18460
Series (1)
GSE69206 Comparing effects of perfusion and hydrostatic pressure on human chondrocytes using gene profiles
BioSample SAMN03734243
SRA SRX1037993

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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