|
| Status |
Public on Apr 30, 2009 |
| Title |
Bmi1 wildtype MEF |
| Sample type |
RNA |
| |
|
| Source name |
Bmi1 wildtype mouse embryonic fibroblast cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
gender: Male
reference: Jacobs, J.J. et al. Nat.Genetics. 2000
antibody: anti-uH2A(E6C5)
|
| Treatment protocol |
None
|
| Growth protocol |
TBX2 immortalized cells were cultured in DMEM supplemented with 10% FBS. Cells were split 1:5 every three days using 0.01% Trypsin-EDTA for the duration of culturing.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Qiagen RNeasy Kit by manufacturers protocol for extraction from animal cells.
|
| Label |
Biotin
|
| Label protocol |
7 ug of total RNA was used to synthesize cDNA. A custom cDNA kit from Life Technologies was used with a T7-(dT)24 primer for this reaction. Biotinylated cRNA was then generated from the cDNA reaction using the BioArray High Yield RNA Transcript Kit. The cRNA was then fragmented in fragmentation buffer (5X fragmentation buffer: 200mM Tris-acetate, pH8.1, 500mM KOAc, 150mM MgOAc) at 94oC for 35 minutes before the chip hybridization. 15 ug of fragmented cRNA was then added to a hybridization cocktail (0.05 ug/ul fragmented cRNA, 50 pM control oligonucleotide B2, BioB, BioC, BioD, and cre hybridization controls, 0.1 mg/ml herring sperm DNA, 0.5 mg/ml acetylated BSA, 100mM MES, 1M [Na+], 20mM EDTA, 0.01% Tween 20).
|
| |
|
| Hybridization protocol |
10 ug of cRNA was used for hybridization. Arrays were hybridized for 16 hours at 45oC in the GeneChip Hybridization Oven 640. The arrays were washed and stained with R-phycoerythrin streptavidin in the GeneChip Fluidics Station 400.
|
| Scan protocol |
the arrays were scanned with the Hewlett Packard GeneArray Scanner. Affymetrix GeneChip Microarray Suite 5.0 software was used for washing, scanning, and basic analysis. Sample quality was assessed by examination of 3’ to 5’ intensity ratios of certain genes.
|
| Description |
Expression study was performed in conjunction with genome-wide localization of ubiquitylated H2A.
|
| Data processing |
MAS5 algorithm was used for data generation.
|
| |
|
| Submission date |
Apr 16, 2009 |
| Last update date |
Apr 30, 2009 |
| Contact name |
Eric M. Kallin |
| E-mail(s) |
eric.kallin@crg.es
|
| Organization name |
Center for Genomic Regulation
|
| Department |
Differentiation and Cancer
|
| Lab |
Thomas Graf
|
| Street address |
C/ Dr. Aiguader, 88
|
| City |
Barcelona |
| State/province |
Barcelona |
| ZIP/Postal code |
08003 |
| Country |
Spain |
| |
|
| Platform ID |
GPL1261 |
| Series (2) |
| GSE15715 |
Gene expression changes in Bmi1 knock-out MEFs as compared to wild-type. |
| GSE15909 |
Gene expression and UH2A ChIP-Seq binding analysis in Bmi1 knock-out and wild type MEFs |
|
