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Sample GSM1162379 Query DataSets for GSM1162379
Status Public on Jun 01, 2015
Title HD291 line cells after 15 mechanic passages
Sample type genomic
 
Source name hESC in mechanic passaging condition
Organism Homo sapiens
Characteristics cell line source gender: male
cell line: HD291
passage number: p13+MP15
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+MP15
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

Data table header descriptions
ID_REF
VALUE CN STATE
CHROMOSOME
POSITION
LOG2 RATIO
SMOOTH SIGNAL
LOH
ALLELE DIFFERENCE

Data table
ID_REF VALUE CHROMOSOME POSITION LOG2 RATIO SMOOTH SIGNAL LOH ALLELE DIFFERENCE
CN_473963 2.000000 1 61723 -0.544128 1.474081 nan nan
CN_473964 2.000000 1 61796 -0.067765 1.474095 nan nan
CN_473965 2.000000 1 61811 -0.013875 1.474098 nan nan
CN_473981 2.000000 1 62908 -0.624313 1.474312 nan nan
CN_473982 2.000000 1 62925 0.183898 1.474316 nan nan
CN_497981 2.000000 1 72764 -0.353801 1.476551 nan nan
CN_502615 2.000000 1 85924 -0.377431 1.480476 nan nan
CN_502613 2.000000 1 85986 0.001945 1.480497 nan nan
CN_502614 2.000000 1 86312 -0.433836 1.480609 nan nan
CN_502616 2.000000 1 86329 -0.689499 1.480614 nan nan
CN_502843 2.000000 1 98590 0.293410 1.485336 nan nan
CN_466171 2.000000 1 228694 0.917943 3.146034 nan nan
CN_468414 2.000000 1 229063 -0.117449 3.146091 nan nan
CN_468412 2.000000 1 229146 0.315430 3.146103 nan nan
CN_468413 2.000000 1 229161 0.177981 3.146106 nan nan
CN_470565 2.000000 1 229607 0.385015 3.146174 nan nan
CN_468424 2.000000 1 235658 0.603996 3.147113 nan nan
CN_468425 2.000000 1 235716 0.261117 3.147122 nan nan
CN_460512 2.000000 1 356431 -0.254976 2.585917 nan nan
CN_460513 2.000000 1 356530 0.667035 2.585919 nan nan

Total number of rows: 1812237

Table truncated, full table size 114261 Kbytes.




Supplementary file Size Download File type/resource
GSM1162379_hff_Mech_20110514_GenomeWideSNP_6_.CEL.gz 27.9 Mb (ftp)(http) CEL
GSM1162379_hff_Mech_20110514_GenomeWideSNP_6_.CN5.CNCHP.gz 34.9 Mb (ftp)(http) CNCHP
Processed data included within Sample table
Processed data provided as supplementary file

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