|
| Status |
Public on Jun 01, 2015 |
| Title |
HD291 line after 30 mechanic passages |
| Sample type |
RNA |
| |
|
| Source name |
hESC in mechanic passaging condition
|
| Organism |
Homo sapiens |
| Characteristics |
cell line source gender: male
cell line: HD291
cell type: Human embryonic stem cells (hESCs)
|
| Treatment protocol |
Mechanical passaging was carried out under an inverted microscope under the hood using scalpels (Assou et al., 2009; Bai et al., 2011). For enzymatic single cell passaging, the cells were pre-treated with Y-27632 for 1h and dissociated with TrypLE™ Select (Invitrogen) for 10 mn at 37C. The reaction was stopped by hESC culture media and single hESC were released by pipetting and passed through a 20 µm strainer to eliminate hFF feeders, then counted and seeded on newly prepared feeder in density of 20 000 cells/cm2 at beginning and progressively to 3 000 cells/cm2 when cells were adaptated to enzymatic passaging. Both mechanic and enzymatic passaging were performed weekly. Experiments on HD291, HD129 and HS306 lines were initiated at p13, p16 and p25 respectively (termed initiating passages) and maintained independently using mechanic passaging (+MP) or enzyamatic passaging (+EP). All cell cultures were regularly checked for mycoplasma contamination and remained negative throughout the experiments.
|
| Growth protocol |
These lines were maintained in culture media consisting of 80% KO-DMEM, 20% KOSR, 2 mM L-glutamine, 1% non-essential amino acids, 0.5 mM β-mercaptoethanol (all from Gibco Invitrogen, Cergy-Pontoise, France) and complemented with 10 ng/mL bFGF (Abcys, Paris, France). These cells were maintained in 35-mm dishes with pre-coated irradiated (40 Gy) human foreskin fibroblast (hFF) feeders and were either enzymatically or mechanically passaged every week.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
In mechanically passaged culture, the hESC colonies were exscinded carefully using a scalpel. In enzymatic-passaged culture, the singles cells were released as described above. As SSEA4 is one of most obstinate pluripotence markers (Ramirez et al., 2011), hESC cells were purified and enriched using the magnetic beads Dynabeads® SSEA-4 (Invitrogen) and the manufacture’s instructions were rigorously followed. The quality control of the purification showed high efficiency and no over-selection of SSEA4+ sub-population. Total RNA was purified from cell culture using the RNeasy Mini Kit (Qiagen, Courtaboeuf, France) following manufacture’s instructions. We added an RNAse-Free DNase step in RNA extraction and an RNsae A treatment in DNA extraction to eliminate residual DNA or RNA.
|
| Label |
biotin
|
| Label protocol |
cRNA synthesis was made using 3`IVT Express kit (Affymetrix) following manufacterer's instructions and starting with 200ng of total RNA.
|
| |
|
| Hybridization protocol |
Labeled cRNA was fragmented in fragmentation buffer (5x buffer:200 mM Tris-acetate (pH 8.1)/500nM KOAc/150 mM MgOAc) before hybridization on the array. Fragmented cRNA was hybridized to the microarrays in 90ul hybridization solution.
|
| Scan protocol |
Human Genome U219 array plate was processed in Gene Atlas according to manufacturer's instructions.
|
| Description |
HD291_p13+M30
Gene expression data from human embryonic cell line HD291 after 30 mechanic passages + initial passages 13 mechanic passages
|
| Data processing |
The data were analyzed with RMA method in Expression Console (Affymetrix).
|
| |
|
| Submission date |
Jun 13, 2013 |
| Last update date |
Jun 01, 2015 |
| Contact name |
Qiang BAI |
| E-mail(s) |
qiang.bai@uliege.be
|
| Phone |
+32 (0)4 366 9841
|
| Organization name |
University of Liege
|
| Department |
GIGA Institute
|
| Lab |
Immunophysiology Laboratory
|
| Street address |
Avenue de l'Hopital 1
|
| City |
Liège |
| ZIP/Postal code |
4000 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL13667 |
| Series (2) |
| GSE47916 |
Expression of four hESC lines in enzymatic and mechanic passaging methods |
| GSE47917 |
Genome alterations in human pluripotent stem cells as very early events highly dependent on cell passaging conditions |
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