|
| Status |
Public on Mar 13, 2009 |
| Title |
small_intestine_3days_after_conditional_Ascl2_deletion_ko_vs_wildtype |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
epithelial cells from small intestine
|
| Organism |
Mus musculus |
| Characteristics |
epithelial intestinal cells of Ah-Cre/Ascl2floxed/wt control animals
|
| Treatment protocol |
For the stem cell signature we used cell fractions of intestines from Lgr5-EGFP-ires-CreERT2 mice, expressing GFP under the control of the Lgr5 promoter. RNA was isolated from two FACS sorted cell populations, one expressing GFP at high levels (GFPhi) and the other expressing GFP at low levels (GFPlo). For the analysis of Ascl2 target genes RNA was isolated from intestinal epithelial cells of Ah-Cre/Ascl2floxed/floxed animals and Ah-Cre/Ascl2floxed/wt control animals 3 and 5 days post induction
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using Trizol (Invitrogen)
|
| Label |
Cy3
|
| Label protocol |
500 ng of total RNA was labeled using low RNA Input Linear Amp kit (Agilent Technologies, Pato Alto, CA, USA).
|
| |
|
| Channel 2 |
| Source name |
epithelial cells from small intestine 3 days after conditional knockout of Ascl2
|
| Organism |
Mus musculus |
| Characteristics |
epithelial intestinal cells from Ah-Cre/Ascl2floxed/floxed animals
|
| Treatment protocol |
For the stem cell signature we used cell fractions of intestines from Lgr5-EGFP-ires-CreERT2 mice, expressing GFP under the control of the Lgr5 promoter. RNA was isolated from two FACS sorted cell populations, one expressing GFP at high levels (GFPhi) and the other expressing GFP at low levels (GFPlo). For the analysis of Ascl2 target genes RNA was isolated from intestinal epithelial cells of Ah-Cre/Ascl2floxed/floxed animals and Ah-Cre/Ascl2floxed/wt control animals 3 and 5 days post induction
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using Trizol (Invitrogen)
|
| Label |
Cy5
|
| Label protocol |
500 ng of total RNA was labeled using low RNA Input Linear Amp kit (Agilent Technologies, Pato Alto, CA, USA).
|
| |
|
| |
| Hybridization protocol |
according to Agilent guidelines
|
| Scan protocol |
according to Agilent guidelines
|
| Description |
total RNA isolated from small intestine 3 days after conditional knockout of Ascl2 hybridized against wildtype tissue, dyeswap
|
| Data processing |
Microarray signal and background information were retrieved using featuere extraction (V.9.5.3, Agilent Technologies). All data analyses were performed using ArrayAssist (5.5.1, Stratagene Inc, La Jolla, CA, USA) and Microsoft Excel (Microsoft Corporation, Redmond, WA, USA). Raw signal intensities were corrected by subtracting local background. Negative values were changed into a positive value close to zero (standard deviation of the local background) in order to allow calculation of ratios between intensities for features only present in one sample (GFPhi or GFPlo) or (Ah-Cre/Ascl2floxed/wt or Ah-Cre/Ascl2floxed/floxed). Data were filtered if both (GFPhi and GFPlo) or (Ah-Cre/Ascl2floxed/wt or Ah-Cre/Ascl2floxed/floxed) intensities were changed or if both intensities were less than two times the background signal and normalized using the Loess algorithm.
|
| |
|
| Submission date |
Dec 23, 2008 |
| Last update date |
Mar 13, 2009 |
| Contact name |
Daniel E. Stange |
| E-mail(s) |
daniel.stange@uniklinikum-dresden.de
|
| Organization name |
University Clinic Dresden
|
| Street address |
Fetscherstr. 74
|
| City |
Dresden |
| ZIP/Postal code |
01307 |
| Country |
Germany |
| |
|
| Platform ID |
GPL4134 |
| Series (1) |
| GSE14201 |
Transcription factor Achaete scute-like 2 (Ascl2) controls intestinal stem cell fate |
|
