|
| Status |
Public on Apr 29, 2011 |
| Title |
Bmi1 embryoid body sample_2 (Bmi1_EB_2) |
| Sample type |
RNA |
| |
|
| Source name |
Embryoid body formation (10 days) of Bmi1 transduced embryonic stem cells (CCE ES cells).
|
| Organism |
Mus musculus |
| Characteristics |
strain: 129/Sv
|
| Treatment protocol |
CCE ES cells were trypsinized and subjected to embryoid body (EB) assay in bacterial Petri dish. At day 10 EBs were harvested and total RNA was isolated.
|
| Growth protocol |
CCE ES cells were cultured on gelatin coated dish in DMEM medium with 15% FSC and leukemia inhibitory factor (LIF). Lentivirus containing mouse BMI1 cDNA was used to infect CCE ES cells in the presence of 8 microgram polybrene. Empty vector served as control. After two passages eGFP positive CCE ES cells were sorted by flow cytometry and cells were further grown in culture medium as above. For differentiation CCE ES cells were subjected to EB assay (10 days).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using RNeasy Mini kit (Qiagen, Hilden, Germany) with DNAse digestion using the maufacturer's buffers and protocols. RNA quality was assessed by Agilent 2100 Bioanalyzer System.
|
| Label |
biotin
|
| Label protocol |
Probe preparation was with 1 microgram RNA using the Affymetrix One-Cycle Target Labeling Kit according to the manufacturer's instructions. Labeled RNA was fragmented by heating to 94°C for 35 minutes in Fragmentatin buffer of the compnay.
|
| |
|
| Hybridization protocol |
Affymetrix arrays were prehybridized with 200 microliter MES hybridization buffer for 10 minutes at 45˚C with rotation (60 U/min) in an Affymetrix Hybridization Oven 640. After removing the prehybridization solution, 10 microgram of labeled cRNA in 200 microliter MES hybridization buffer were applied to the array leaving a small air bubble. Arrays were incubated overnight at 45˚C with rotation (60U/min) in the hybridization chamber.
|
| Scan protocol |
Arrays were scanned using Affymetrix GeneChip Scanner 3000.
|
| Description |
BioConductor software package (Gentleman, R. C., Carey, V. J., Bates, D. M. et al. Bioconductor: Open software development for computational biology and bioinformatics. Genome Biol. 2004; 5: R80) (www.bioconductor.org).
|
| Data processing |
CEL files were processed using the BioConductor software package (www.bioconductor.org). Normalization was done employing RMA (Irizarry et al., Biostatistics 4, 249-264, 2003).
|
| |
|
| Submission date |
Mar 19, 2010 |
| Last update date |
Apr 30, 2011 |
| Contact name |
Martin Zenke |
| E-mail(s) |
[email protected]
|
| Phone |
+49-241-80 80760
|
| Organization name |
Institute for Biomedical Engineering
|
| Department |
Cell Biology
|
| Street address |
Universitatsklinikum Aachen, RWTH
|
| City |
Aachen |
| State/province |
NRW |
| ZIP/Postal code |
52074 |
| Country |
Germany |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE20958 |
Polycomb Group Protein Bmi1 Promotes Hematopoietic Cell Development from ES Cells |
|
