|
| Status |
Public on May 28, 2020 |
| Title |
05_341_WT-Low |
| Sample type |
RNA |
| |
|
| Source name |
WT-Low
|
| Organism |
Mus musculus |
| Characteristics |
genotype/variation: WT
cell type: CD24(low)/EGFP(Low) transit amplifying population
|
| Treatment protocol |
Mice treated with tamoxifen for five consecutive days and sacrificed 1 month after treatment. Intestinal tissue was incubated in 15 mM EDTA in PBS for 25 minutes on ice then vigorously vortexed to release crypts. Crypts were dissociated by incubation in dispase buffer containing 0.3 U/ml dispase (Corning), 0.8 U/ml DNase (Ambion), 10 µM Y-27632 (Enzo) at 37°C for 30 min. Intestinal stem cells and their direct transit amplifying progenitors from individual mice were enriched by sorting CD24(low)/EGFP(High) or CD24(low)/EGFP(Low) populations, respectively.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were sorted directly into lysis buffer and RNA was extracted using the RNeasy Micro Kit (Qiagen).
|
| Label |
biotin
|
| Label protocol |
1 ng of total RNA is reverse transcribed following the Ovation Pico System V2 (Tecan). Briefly, the resulting double strand cDNA is used for amplification based on SPIA technology. After purification according to Nugen protocol, 5.5 ug of Sens Target DNA are fragmented and biotin labelled using Encore Biotin Module kit (Tecan).
|
| |
|
| Hybridization protocol |
After control of fragmentation using Bioanalyzer 2100, cDNA is then hybridized to GeneChip® MouseGene2.0 ST (Affymetrix) at 45°C for 17 hours.
|
| Scan protocol |
After overnight hybridization, chips are washed on the Fluidic Station FS450 following specific protocols (Affymetrix) and scanned using the GCS3000 7G.
|
| Description |
WT sorted EGFP(Low) transit amplifying population.
|
| Data processing |
The scanned images are then analyzed with Expression Console software (Affymetrix) to obtain raw data (cel files) and metrics for Quality Controls. The observations of some of these metrics and the study of the distribution of raw data show no outlier experiment.
RMA normalization is performed using R with Version 21 of EntrezGene CDF brainarray.
|
| |
|
| Submission date |
Sep 05, 2019 |
| Last update date |
May 28, 2020 |
| Contact name |
Franck Letourneur |
| E-mail(s) |
franck.letourneur@inserm.fr
|
| Organization name |
INSERM
|
| Department |
U1016
|
| Lab |
genomic
|
| Street address |
22 rue mechain
|
| City |
Paris |
| ZIP/Postal code |
75014 |
| Country |
France |
| |
|
| Platform ID |
GPL23092 |
| Series (1) |
| GSE136952 |
Autophagy maintains intestinal stem cell integrity |
|
