|
| Status |
Public on Mar 01, 2014 |
| Title |
24h_co_enriched biol. replicate 1 |
| Sample type |
RNA |
| |
|
| Source name |
NMuMG_untreated_4sU for 2hrs
|
| Organism |
Mus musculus |
| Characteristics |
cell line: NMuMG
cell type: mammary gland cells
treated with: none (untreated control)
incubated with: 4sU for 2hrs (22hrs-24hrs)
molecule subtype: 4sU-labeled RNA enriched from total RNA
|
| Treatment protocol |
Cells were seeded 24hrs prior to treatments. Cells were treated with 5ng/ml TGF-b1 for 24hrs or left untreated. For enrichment of newly-synthesized RNA, cells were treated with 200microM 4sU for the last 2hrs of incubation perios with TGF-b1 (22hrs-24hrs)
|
| Growth protocol |
Cells were grown under standard cell culture conditions (37◦C, 5%CO2) in ATCC DMEM-media, 10%FCS, 10microgr/ml insulin)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using RNeasy kit (Qiagen) according to manufacturer's instructions, including on-column DNase-digest . 4sU-labeled RNA was extracted from respective total RNA fractions using custom-made mercury-based columns (Kenzelmann et al. 2007)
|
| Label |
biotin
|
| Label protocol |
Total and 4sU-labeled/enriched RNA were amplified via GeneChip® 2-Cycle cDNA synthesis kit according to instructions (Affymetrix) and transcibed into cRNA overnight using MegaScript® kit (Ambion). Purified cRNA was reverse-transcribed into cDNA. After clean-up of cDNA, biotin-labelled cRNA was generated using GeneChip™ IVT labelling ki. 5microgr. of biotinylated cRNA was purified and fragmented.
|
| |
|
| Hybridization protocol |
Hybridisation onto 430_2.0 mouse arrays was performed according to manufacturer's instructions (16hrs, 45◦C)
|
| Scan protocol |
Arrays were scanned using GeneChip™ scanner 3000.
|
| Description |
24h_co_enriched 1.CEL
Gene expression data from cells not treated with TGF-b1 but incubated with 4sU for 2hrs (22hrs-24hrs)
|
| Data processing |
The data was analyzed using R (v.3.0.2)/Bioconductor (v.2.13) VSN-normalized
|
| |
|
| Submission date |
Feb 06, 2014 |
| Last update date |
Mar 01, 2014 |
| Contact name |
Alexander Feuerborn |
| E-mail(s) |
alexander.feuerborn@path.ox.ac.uk
|
| Organization name |
Sir William Dunn School of Pathology
|
| Street address |
South Parks Road
|
| City |
Oxford |
| ZIP/Postal code |
OX1 3RE |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL18223 |
| Series (2) |
| GSE54716 |
Basonuclin-1 modulates epithelial plasticity and TGF-β1-induced loss of epithelial cell integrity [NIAC-NTR] |
| GSE54717 |
Basonuclin-1 modulates epithelial plasticity and TGF-β1-induced loss of epithelial cell integrity |
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