|
| Status |
Public on May 30, 2012 |
| Title |
OSKM_MEF_Nanog_KO |
| Sample type |
RNA |
| |
|
| Source name |
OSKM_MEF_Nanog_KO
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Mouse Embryonic Fibroblast Cells (MEF) generated from Nanog-/- ESCs (Mitsui et al. Cell 2003)
genotype/variation: transduced with OSKM lentivirus for 8 days
|
| Treatment protocol |
Cells were washed with PBS and then harvested by trypsinization for RNA extraction
|
| Growth protocol |
N2B27 2i/LIF mouse ESC media
|
| Extracted molecule |
total RNA |
| Extraction protocol |
mRNA was extracted byTRIZOL reagent (invitogene) according to manufacturer's instructions
|
| Label |
Biotin
|
| Label protocol |
250 ng total RNA was used to prepare biotinylated aRNA (cRNA) according to the manufacturer’s protocol (Affymetrix 3’ IVT Express Kit). Briefly, total RNA undergoes T7 oligo(dT)-primed reverse transcription to synthesize first-strand cDNA containing a T7 promoter sequence. This cDNA is then converted into a double-stranded DNA template for transcription using DNA Polymerase and RNase H to simultaneously degrade the RNA and synthesize second strand cDNA. In vitro transcription synthesizes aRNA and incorporates a biotin-conjugated nucleotide. The aRNA is then purified to remove unincorporated NTPs, salts, enzymes, and inorganic phosphate. Fragmentation of the biotin-labeled aRNA prepares the sample for hybridization onto GeneChip 3’ expression arrays.
|
| |
|
| Hybridization protocol |
Samples were prepared for hybridization using 12.5 µg of biotinylated aRNA in a 1X hybridization cocktail according the Affymetrix hybridization manual. Additional hybridization cocktail components were provided in the Affymetrix GeneChip Hybridization, Wash and Stain Kit. GeneChip arrays were hybridized in a GeneChip Hybridization Oven at 45°C for 16 hours at 60 RPM. Washing was done using a GeneChip Fluidics Station 450 according to the manufacturer’s instructions, using the buffers provided in the Affymetrix GeneChip Hybridization, Wash and Stain Kit.
|
| Scan protocol |
Arrays were scanned on a GeneChip Scanner 3000 and images were extracted with Affymetrix GeneChip Command Console (AGCC), and analyzed using GeneChip Expression Console.
|
| Description |
Gene expression data of the indicated cell line
|
| Data processing |
The data were analyzed with Affy Expression Console software, using RMA method. Probes were further filtered to include only probes that have at least one call > 6 (in log2-scale)
|
| |
|
| Submission date |
May 29, 2012 |
| Last update date |
May 30, 2012 |
| Contact name |
Noa Novershtern |
| E-mail(s) |
noa.novershtern@weizmann.ac.il
|
| Organization name |
Weizmann Institute of Science
|
| Department |
Molecular Genetics
|
| Street address |
Weizmann Institute
|
| City |
Rehovot |
| ZIP/Postal code |
7610001 |
| Country |
Israel |
| |
|
| Platform ID |
GPL6246 |
| Series (2) |
| GSE35775 |
The H3K27 demethylase Utx facilitates somatic and germ cell epigenetic reprogramming to pluripotency [Affymetrix gene expression] |
| GSE37822 |
The H3K27 demethylase Utx facilitates somatic and germ cell epigenetic reprogramming to pluripotency |
|
