|
| Status |
Public on Jul 04, 2024 |
| Title |
SCWAT - ChREBPtotal_KO - Rep4 |
| Sample type |
RNA |
| |
|
| Source name |
Subcutaneous White Adipose Tissue
|
| Organism |
Mus musculus |
| Characteristics |
strain: ChREBPtotal
genotype: ChREBPtotal_KO
washbatch: 3
|
| Treatment protocol |
All animal were submitted to a 24h fasting period followed by 18h refeeding with food and 20% glucose-supplemented water to favor ChREBP activation.
|
| Growth protocol |
Mice were fed a regular diet and housed in regular condition of temperature and dark-ligt cycles.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tissues were homogenized in Qiazol buffer (Qiagen 79306) using Precellys tissue homogenizer. Total RNA from tissues was extracted using RNeasy kit (Qiagen 75162).
|
| Label |
Cy3
|
| Label protocol |
For each sample, Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng of total RNA using the One-Color Quick Amp Labeling kit (Agilent Technologies, Santa Clara, CA) according to the manufacturer's instructions, followed by Agencourt RNAClean XP (Agencourt Bioscience Corporation, Beverly, Massachusetts) purification. Dye incorporation and cRNA yield were checked using Dropsense™ 96 UV/VIS droplet reader (Trinean, Belgium).
|
| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity >6 pmol Cy3/µg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 µl containing 10x Agilent fragmentation buffer and 25x Agilent blocking agent following the manufacturer's instructions (Agilent Technologies, Santa Clara, CA). On completion of the fragmentation reaction, 25 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to SurePrint G3 Mouse GE v2 microarray (8X60K, Design 074809 enclosed in Agilent SureHyb-enabled hybridization chambers for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed sequentially in Wash buffer 1 (Agilent Technologies, 1 min) and Wash buffer 2 (Agilent Technologies, 37°C, 1 min).
|
| Scan protocol |
Slides were scanned immediately after washing on a Agilent G2505C Microarray Scanner with Agilent Scan Control A.8.5.1 software
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.10.1.1 (Agilent Technologies, Santa Clara, CA) using default parameters (protocol GE1_1010_Sep10 and Grid: 074809_D_F_20150624). All subsequent data analyses were done under R (www.r-project.org) using packages of Bioconductor (www.bioconductor.org). Raw data (median of pixels intensity) were imported into R using the read.maimages function from the limma package with the following weight function (assigning a weight of 1 or 0 to each spot): myfunw<-function(x) {okType<-x$ControlType==0; okFoundGreen<-x$gIsFound==1; okPos=x$gIsPosAndSignif==1; okWellAbove<- x$gIsWellAboveBG==1; as.numeric(okType & okFoundGreen & okPos & okWellAbove);} We selected the spots with a minimal weight of 1 for 21 out of 26 microarrays or with a minimal weight of 5 per group from at least one experimental group. At this step, 42868 spots out of 62976 were selected. Data were then stored in an ExpressionSet object and normalized by the quantile method using the normalize.quantiles function from the preprocessCore R library. Replicated probes on the array (identical ProbeName) were resolved by taking the median normalized signal of each set of replicated probes. The resulting matrix has 40168 rows each corresponding to a unique ProbeName (provided as data Matrix).
|
| |
|
| Submission date |
Jul 06, 2021 |
| Last update date |
Jul 04, 2024 |
| Contact name |
Dominique Langin |
| E-mail(s) |
dominique.langin@inserm.fr
|
| Organization name |
INSERM
|
| Lab |
I2MC
|
| Street address |
1 avenue du Professeur Jean Poulhès
|
| City |
Toulouse Cedex 4 |
| ZIP/Postal code |
31432 |
| Country |
France |
| |
|
| Platform ID |
GPL21163 |
| Series (2) |
| GSE179563 |
Transcriptomic analyses of ChREBPtotal-null mice and ChREBPβ-null mice subcutaneous inguinal white adipose tissue |
| GSE179564 |
Transcriptomic analyses of ChREBPtotal-null mice and ChREBPβ-null mice |
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