|
| Status |
Public on Nov 06, 2019 |
| Title |
Early passage of MEF cells derived from Itpr2 Knock-out mice replicat 2 |
| Sample type |
RNA |
| |
|
| Source name |
Early passage Itpr2 KO MEFs
|
| Organism |
Mus musculus |
| Characteristics |
genotype: Itpr2 KO
passage: Early
|
| Treatment protocol |
MEFs were cultured during 3 passages (Early passage) or 5 passages (Late passage)
|
| Growth protocol |
Mouse Embryonic Fibroblasts (MEFs) were prepared with embryos at E12.5 and cultured in Dulbecco′s modified Eagle′s medium with GlutaMax, 10% FBS, 1% penicillin/streptomycin and 1% of Gibco™ MEM Non-Essential Amino Acids Solution (ThermoFisher Scientific).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from MEFs in culture using Nucleospin RNA kit (Macherey Nagel)
|
| Label |
Cy3
|
| Label protocol |
cRNAs were synthesized and labeled with the Cy3 dye from 100 ng of total RNA using the one-color Low Input Quick Amp Labeling Kit (Agilent Technologies) following the manufacturer's recommandations, followed by RNeasy Mini column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. After fragmentation 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent GE Whole Human Genome Oligo Microarrays (Agilent-026652) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried briefly in acetonitrile.
|
| Scan protocol |
Microarrays were scanned with an Agilent DNA microarray scanner G2565CA (Agilent Technologies).Fluorescent signals were extracted and normalized with the Feature Extraction software version 10.5.1.1 (Agilent Technologies),
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities.
|
| |
|
| Submission date |
Nov 05, 2019 |
| Last update date |
Nov 06, 2019 |
| Contact name |
jean-michel flaman |
| E-mail(s) |
jean-michel.flaman@lyon.unicancer.fr
|
| Phone |
+33687477812
|
| Organization name |
Inserm 1052
|
| Department |
CRCL
|
| Street address |
28 rue Laennec
|
| City |
Lyon |
| ZIP/Postal code |
69373 |
| Country |
France |
| |
|
| Platform ID |
GPL11202 |
| Series (1) |
| GSE139982 |
Transcriptome profiling of early or late passage MEFs derived from calcium channel ITPR2 KO mice |
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