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| Status |
Public on Oct 24, 2016 |
| Title |
Low HG - H9 cells cultured in 80% mebil gel for 7days with mTeSR1 medium, rep 3 |
| Sample type |
RNA |
| |
|
| Source name |
H9 cells cultured in 80% mebil gel for 7days with mTeSR1 medium.
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: embryonic stem cell
tissue: in vitro
|
| Treatment protocol |
The dissociated hPSCs were resuspended in mTeSR-1 medium mixed with different concentrations of PNIPAAm-β-PEG hydrogel (HG) (Mebiol Inc., Hiratsuka, Japan) supplemented with 10 µM Y-27632. HG concentrations of 61 and 76 mg ml-1 were utilized to obtain cellular matrices with stiffness properties identified as “Low HG” and “HG”, respectively.
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| Growth protocol |
H9 human embryonic stem cells (hESCs) were cultured in mTeSR-1 medium (Stem Cell Technology) supplemented with 10 µM Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor Y-27632 (Wako Pure Chemical Industries, Osaka, Japan) on day 1, and the medium was changed daily.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from the cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer recommended protocol. After culturing hPSCs in the microfluidic device, the system was placed on ice and the cells were transferred into a 1.5-mL tube. The cells were then allowed to settle to the bottom of the tube with gravity. After centrifuging for 5 min at 300 xg, the supernatants were aspirated to remove the culture medium and trypsin. The cell pellet was then treated with cell lysis buffer from the RNeasy Mini Kit. After homogenizing the lysates, 70% ethanol was added, and the mixture was applied to a spin column in a tube, and centrifuged at 8,000 xg for 15 sec. The spin column was rinsed with a washing buffer. Finally, RNase-free water was used to elute total RNA from the spin column. The total concentration of retrieved RNA was quantified using the NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific), and its quality was evaluated with the BioAnalyzer 2100 and RNA 6000 Nano kit (Agilent Technologies, Santa Clara, CA, USA).
|
| Label |
Cy3
|
| Label protocol |
A total of 100 ng of ribonucleic acid (RNA) from each sample was amplified and labeled with cyanine 3 (Cy3) fluorescent dye to synthesize complementary viral RNA (cRNA) using a One-Color, Low Input Quick Amp Labeling Kit (Agilent Technologies, Santa Clara, CA, USA).
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| |
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| Hybridization protocol |
0.6 µg of synthesized cRNA samples were hybridized to SurePrint G3 Human GE 8×60K Ver.2.0 microarrays (Agilent Technologies, Santa Clara, CA, USA).
|
| Scan protocol |
Agilent DNA microarray scanner (G2565CA) was used to obtain DNA microarray images.
|
| Description |
H9 cells cultured in 80% mebil gel for 7days with mTeSR1 medium, rep 3
|
| Data processing |
Data were pre-processed using limma R/Bioconductor package including RMA background correction, quantile normalisation and log2 transformation. Control probes were removed after normalisation. Detection threshold (log2) for Stemformatics graphing uses 95th percentile of negative control probe expression as per limma recommendation. Median expression is median (log2) of above-threshold expression of all non-control probes.
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| |
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| Submission date |
Apr 15, 2016 |
| Last update date |
Feb 27, 2019 |
| Contact name |
Othmar Korn |
| Organization name |
Australian Institute for Bioengineering and Nanotechnology
|
| Street address |
University of Queensland
|
| City |
St. Lucia |
| State/province |
Queensland |
| ZIP/Postal code |
4067 |
| Country |
Australia |
| |
|
| Platform ID |
GPL17077 |
| Series (1) |
| GSE80324 |
Expression profiling of cell growth on different layers |
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