|
| Status |
Public on May 07, 2012 |
| Title |
2cell embryo_matRing1/Rnf2 double mutant_no treatment_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
2-cell embryo, matRing1/Rnf2 double mutant, no treatment
|
| Organism |
Mus musculus |
| Characteristics |
strain: mixed 129/Sv and C57BL/6J
genotype: maternal Ring1-Rnf2-
developmental stage: 2-cell embryo
treatment: none
|
| Treatment protocol |
For inhibiting de novo transcription, IVF embryos were transferred to KSOM containing 24 µg/ml α-amanitin at 6 hpf and harvested for expression profiling at 35 hpf.
|
| Growth protocol |
Mouse oocytes were derived from superovulated 5- to 10-week-old females according to standard procedures. Metaphase II (M-II)-arrested eggs were collected from PMSG- and hCG-primed (5 U each, Intervet) mice 14 h after hCG injection and fertilized in vitro by wild type sperm. Sperm was obtained from 10-16 week old control males. Sperm capacitation was carried out in human tubular fluid (HTF) containing 9 mg/ml BSA for 2h. IVF was performed in capacitation medium for 2 h, and thereafter, the embryos were cultured in KSOM medium plus amino acids (Chemicon) in a humidified atmosphere of 5% CO2 in air until required. For inhibiting de novo transcription, IVF embryos were transferred to KSOM containing 24 µg/ml α-amanitin at 6 hpf and harvested for expression profiling at 35 hpf.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
2-cell embryos were pooled from several mice and RNA was isolated from batches of 40 oocytes, 3 (or 2) biological replicates per genotype/treatment. RNA was isolated using the PicoPureTM RNA Isolation Kit (KIT0202) according to the manufacturer’s instructions (Stratagene). The quality of the RNA was assessed using the Agilent 2100 Bioanalyzer and RNA 6000 Pico Chip. The extracted RNA was converted into OmniPlex WTA cDNA libraries and amplified by WTA PCR using reagents supplied with the TransPlex Whole Transcriptome Amplification kit (WTA1, Sigma, USA) following the manufacturer’s instructions with minor modifications. The obtained cDNA was purified using the GeneChip cDNA Sample Cleanup Module (Affymetrix).
|
| Label |
Biotin
|
| Label protocol |
RNA was processed according to the GeneChip Whole Transcript (WT) Double-Stranded Target Assay Manual from Affymetrix (GeneChip Whole Transcription Sense Target Labeling technical manual, Rev. 2).
|
| |
|
| Hybridization protocol |
Affymetrix GeneChip arrays were hybridized following the GeneChip Whole Transcript (WT) Sense Target Labeling Assay Manual (Affymetrix) with a hybridization time of 16h. The Affymetrix Fluidics protocol FS450_0007 was used for washing.
|
| Scan protocol |
Scanning was performed with Affymetrix GCC Scan Control v. 3.0.0.1214 on a GeneChip® Scanner 3000 with autoloader.
|
| Description |
2cell_matRing1-/Rnf2- dm_no treatment_1
|
| Data processing |
Raw data was read into R (version 2.10.0) with the ReadAffy() command of the Bioconductor (version 2.5) package affy(). The data was normalized and probeset-level values calculated with the vsnrma() function of the vsn package. For probeset mapping and annotation the CDF environment MoGene-1_0-st-v1.r3.cdf (as provided by Bioconductor) and annotation from Netaffx (www.netaffx.com) (as provided in the attached file) were used.
|
| |
|
| Submission date |
Apr 19, 2011 |
| Last update date |
May 08, 2012 |
| Contact name |
Antoine Peters |
| E-mail(s) |
[email protected]
|
| Organization name |
Friedrich Miescher Institute for Biomedical Research (FMI)
|
| Street address |
Maulbeerstrasse 66
|
| City |
Basel |
| ZIP/Postal code |
4058 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL6246 |
| Series (2) |
| GSE28710 |
Polycomb function during oogenesis is required for mouse early embryonic development (2-cell embryos) |
| GSE28711 |
Polycomb function during oogenesis is required for mouse early embryonic development |
|
