|
| Status |
Public on Jun 01, 2015 |
| Title |
HD291 line cells after 30 enzymatic and 15 mechanic passages |
| Sample type |
genomic |
| |
|
| Source name |
hESC in swapped enzymatic/mechanic passaging condition
|
| Organism |
Homo sapiens |
| Characteristics |
cell line source gender: male
cell line: HD291
passage number: p13+EP30MP15
|
| 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 37ºC. 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 |
genomic DNA |
| 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. Genomic DNA was purified from cell culture using the QIAamp DNA Mini Kit (Qiagen, Courtaboeuf, France) following manufacture’s instructions. We added an RNsae A treatment in DNA extraction to eliminate residual DNA.
|
| Label |
biotin
|
| Label protocol |
Affymetrix standard procedure
|
| |
|
| Hybridization protocol |
Affymetrix standard procedure
|
| Scan protocol |
Affymetrix standard procedure
|
| Description |
HD291_p13+EP30MP15
|
| Data processing |
The genotyping and copy number variation result were processed with Genotype Console (Affymetrix);
affymetrix-algorithm-name/version = CN5 v.5.0.0,
affymetrix-algorithm-param-ArraySet = GenomeWideSNP_6,
program-name = Genotyping Console v.4.0.3566.17491
CNCHP files were results for copy number variation data.
|
| |
|
| Submission date |
Jun 13, 2013 |
| Last update date |
Jun 01, 2015 |
| Contact name |
Qiang BAI |
| E-mail(s) |
qiang.bai@inserm.fr
|
| Phone |
+33 (0)4 67 41 52 08
|
| Organization name |
PhyMedExp INSERM U1046
|
| Lab |
Maladies respiratoires et environnement
|
| Street address |
371 Avenue du Doyen Gaston Giraud
|
| City |
Montpellier |
| ZIP/Postal code |
34295 |
| Country |
France |
| |
|
| Platform ID |
GPL6801 |
| Series (2) |
| GSE47914 |
Genomic instability of human embryonic stem cells in mechanic and enzymatic passaging culture |
| GSE47917 |
Genome alterations in human pluripotent stem cells as very early events highly dependent on cell passaging conditions |
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