|
| Status |
Public on Oct 29, 2009 |
| Title |
reprogrammed epiblast ES-like cells passage 4 rEpiES#3 |
| Sample type |
RNA |
| |
|
| Source name |
reprogrammed epiblast ES-like cells passage 4
|
| Organism |
Mus musculus |
| Characteristics |
cell type: reprogrammed epiblast ES-like cells
age: passage 4
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to manufacturer's instructions
|
| Label |
digoxigenin
|
| Label protocol |
Digoxigenin labeled cRNA was prepared according to Applied Biosystems nanoamp RT-IVT protocol
|
| |
|
| Hybridization protocol |
Hybridization was performed according to Applied Biosystems Chemiluminescence Detection Kit Protocol (PN 4339627) using 10 ug fragmented cRNA
|
| Scan protocol |
Image was captured using CCD camera came with AB1700 Microarray Analyzer
|
| Description |
epiblast from e5.5-e7.5 postimplantation embryos obtained as described above, was dissected with glass needles based on morphology, and treated with egta in pbs for 10min, followed by trypsin for 5min at room temperature. epiblast cells were then dissociated into single cells by pipeting with glass capillary. these of epiblast cells were cultured in ‘es’ medium (dmem/f12 medium plus 20% fetal bovine serum and 1000u/ml lif) on mitomycin treated mouse embryonic fibroblast (mefs) feeder cells. these epiblast cells grew efficiently as epithelial colonies after 4-5 days culture. the resulting colonies were treated with 1mg/ml collagenase for 10min at room temperature and picked, dissociated into smaller clumps by mechanically cutting them into smaller pieces using glass needles, and similarly passaged at every 5-6 days intervals. these referred to as primary repi cell lines, could be passaged extensively in es medium, following treatment with collagenase and mechanical dissociation. the morphology of repi colonies was overall similar to that of episcs and not es cells; they were flatter and bigger than es colonies and positive for alkaline phosphatase, but were negative for oct4-gfp expression. following culture of repi cell lines for 14-35 days, a few gfp-positive cells appeared in repi colonies. the clones bearing gfp-positive cells were isolated using glass needles, and passaged following treatment with collagenase and dissociated mechanically. when these colonies were large enough with gfp positive cells, they were treated with trypsin which on subsequent culture produced gfp positive colonies. we refer to these cells as repies cells. these repies have so far been propagated for more than 40 passages (1: 10 to 1:20 dilution each passage). The number of cell lines we derived from postimplantation embryos is summarized.
Gene expression data from mouse embyronic cells using Applied Biosystems AB1700 Whole Genome Microarray
|
| Data processing |
Quantile normalization was performed. The normalization was performed using ABarray bioconductor package
|
| |
|
| Submission date |
Mar 31, 2009 |
| Last update date |
Oct 29, 2009 |
| Contact name |
Fuchou Tang |
| Organization name |
University of Cambridge
|
| Department |
Wellcome Trust/Cancer Research UK Gurdon Institute
|
| Street address |
Tennis Court Road
|
| City |
Cambridge |
| State/province |
Cambridgeshire |
| ZIP/Postal code |
CB2 1QN |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL2995 |
| Series (1) |
| GSE15487 |
Breaking the epigenetic barrier during reversion of post-implantation epiblast cells |
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