|
| Status |
Public on Sep 01, 2013 |
| Title |
Fibroblast_clone_embryonic_E6.5_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
E6.5 tailtip fibroblast clone, embryonic
|
| Organism |
Mus musculus |
| Characteristics |
gender: female
donor cell: tail tip fibroblast cell
tissue: embryonic
embryonic day: E6.5
|
| Treatment protocol |
Embryos that reached the 2-cell stage were transferred into the oviducts of ICR recipient mice at day 1 of pseudopregnancy (the day a copulatory plug was observed after mating with a vasectomized male mouse). On day 7 of pregnancy, corresponding to E6.5, the implanted embryos were recovered carefully from the uteri and isolated from decidua using fine forceps and a needle under a dissecting microscope. After incubation in PBS with 0.25% pancreatin and 0.05% trypsin for 10 minutes at 4 °C, embryos were transferred to DMEM containing 10% FBS and then the visceral endoderm layer was detached from epiblast or extraembryonic ectoderm by being gently aspirated through a mouth pipette several times. Thereafter, the epiblast was separated from the extraembryonic ectoderm using a fine tungsten needle. Isolated epiblasts or extraembryonic regions (extraembryonic ectoderm and ectoplacental cone) were subjected to microarray gene analyses.
|
| Growth protocol |
In vitro fertilized (IVF) or cloned embryos were cultured in potassium modified simplex optimization medium (KSOM) at 37.5 ºC in a humidified incubator with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with TRIzol (Life Technologies) from epiblasts or extraembryonic regions derived from single embryos and subjected to linear amplification using TargetAmp Two-Round Aminoallyl-aRNA Amplification Kits (Epicentre Biotechnologies).
|
| Label |
Cy3
|
| Label protocol |
5.0 ug of amplified RNA was labelled with Cianine-3 (Cy3) dye (GE Healthcare) for 90 minutes at RT. Labelled RNAs were purified with RNeasy MinElute kit (Qiagen) and checked with the NanoDrop ND-1000 Spectrophotometer
|
| |
|
| Hybridization protocol |
1.65ug Cy3-labelled RNAs were fragmented at 60°C for 30 minutes and hybridized at 65°C for 17 hours according to manufacture's instructions.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting (Scan Area 61x21.6 mm, Scan resolution 5 um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
E6.5 tailtip fibroblast clone, embryonic, replicate2
|
| Data processing |
The scanned images of microarray slides were processed using Feature Extraction software 10.5.1 (Agilent Technologies). All raw data were loaded into Gene Spring GX 12.5 (Agilent Technologies) and transformed by default setting.
|
| |
|
| Submission date |
Jul 24, 2013 |
| Last update date |
Sep 01, 2013 |
| Contact name |
Kimiko Inoue |
| E-mail(s) |
kimiko.inoue@riken.jp
|
| Organization name |
BRC, RIKEN
|
| Department |
Bioresource Engineering Division
|
| Street address |
3-1-1 Koyadai
|
| City |
Tsukuba |
| State/province |
Ibaraki |
| ZIP/Postal code |
305-0074 |
| Country |
Japan |
| |
|
| Platform ID |
GPL7202 |
| Series (2) |
| GSE49171 |
Comparative gene expression analyses using post-implanted E6.5 cloned embryos |
| GSE49173 |
Donor cell type-specific gene expression in embryonic but not extraembryonic tissues of postimplantation embryos cloned from somatic cells |
|
