|
| Status |
Public on Dec 01, 2012 |
| Title |
ES_undifferentiated_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
undifferentiated murine ES cells
|
| Organism |
Mus musculus |
| Characteristics |
developmental stage: undifferentiated murine ES cells
passage number: passage 9
cell line: aPIG D3 ES cells
origin of a cell line: Reference: Kolossov E. et al., J Exp Med 203(10):2315-2327, 2006.
|
| Growth protocol |
αPIG-ES and αPIG-iPS cells were cultured on Mitomycin C-inactivated neomycin resistant murine embryonic fibroblasts in the presence of LIF. αPIG-ES and αPIG-iPS cells were differentiated in a standardized spinner flask system to allow spontaneous differentiation. Briefly, one million undifferentiated ES or iPS cells were suspended in 14 ml of differentiation medium (Iscove’s Modified Dulbecco’s Medium supplemented with 20% FBS, 10 μM 2 mercaptoethanol and 1x non-essential amino acids) and cultured for 2 days in non-adherent plates on a shaker under continuous agitation to allow formation of embryoid bodies (referred to as ES-EB and iPS-EB hereafter). At day 2 of differentiation, the formed EB were counted and diluted into fresh 200 ml medium contained within a sterile spinner flask (Cell spin 250) to a density of 28000 EB per one spinner flask. The differentiation process continued for 6-7 days (without medium change) until the first appearance of green fluorescence in spontaneously beating EBs. On day 9 of differentiation, fresh medium supplemented with puromycin (8 μg/ml) was added to select for pure CM. After 2-3 days of selection, the surviving cardiac clusters were pooled together by removing from spinner. These were then resuspended in fresh medium with puromycin and then incubated in non-adherent culture dishes and cultured for another 5-6 days on the shaker. Fresh medium containing puromycin was replaced every 2 days until pure beating cardiac clusters were obtained. On day 16 of differentiation after 6-8 days of puromycin treatment cardiac clusters were used for RNA isolation using Trizol reagent. RNA was also isolated from the same batch of untreated whole EBs at day 16 of differentiation. Primary murine adult tail tip fibroblasts were established in cultured and used for RNA preparation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from all cell samples was isolated using TRIzol Reagent (Invitrogen).
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared with the Ambion MessageAmp kit for Illumina arrays
|
| |
|
| Hybridization protocol |
Standard Illumina hybridization protocol
|
| Scan protocol |
Standard Illumina scanning protocol
|
| Description |
replicate 2
|
| Data processing |
The data were normalised using quantile normalisation in R
|
| |
|
| Submission date |
Oct 09, 2009 |
| Last update date |
Dec 19, 2012 |
| Contact name |
Joachim Schultze |
| E-mail(s) |
j.schultze@uni-bonn.de
|
| Organization name |
LIMES (Life and Medical Sciences Center Genomics and Immunoregulation)
|
| Department |
Genomics and Immunoregulation
|
| Street address |
Carl-Troll-Strasse 31
|
| City |
Bonn |
| State/province |
NRW |
| ZIP/Postal code |
53115 |
| Country |
Germany |
| |
|
| Platform ID |
GPL6887 |
| Series (1) |
| GSE18514 |
Transcriptomic profiling of murine ES and iPS cells, embryoid bodies, and ES and IPS cell-derived cardiomyocytes |
|
