|
| Status |
Public on Mar 16, 2017 |
| Title |
10 days mouse embryoid body NT, biological rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
10 days mouse embryoid body
|
| Organism |
Mus musculus |
| Characteristics |
cell type: mouse embryoid bodies
genotype/variation: Wildtype control
age: day 10
|
| Growth protocol |
All embryoid bodies were grown for 10 days in suspension in low attachment plates without LIF
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using TRIzol reagent (Invitrogen, 15596-026) according to manufacturer's instructions and purified with an RNeasy mini kit (Qiagen, 74104).1 ug of total RNA was amplified via the aRNA MessageAmp II kit (Ambion, 1751)
|
| Label |
Cy5
|
| Label protocol |
For each hybridization, 300 ng of Cy3 probes and 300 ng of Cy5 probes of amplified cRNA were mixed and added to 5 ul of 10x Blocking Agent, 1 ul of 25x Fragmentation Buffer and Nuclease free water in a 25 ul reaction, incubated at 60ºC for 30 minutes to fragment RNA and stopped with 25 ul of 2x Hybridization Buffer.
|
| |
|
| Channel 2 |
| Source name |
10 days mouse embryoid body. Dido3deltaCT+HADidoNT
|
| Organism |
Mus musculus |
| Characteristics |
cell type: mouse embryoid bodies
genotype/variation: Dido3deltaCT+HADidoNT
age: day 10
|
| Growth protocol |
All embryoid bodies were grown for 10 days in suspension in low attachment plates without LIF
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using TRIzol reagent (Invitrogen, 15596-026) according to manufacturer's instructions and purified with an RNeasy mini kit (Qiagen, 74104).1 ug of total RNA was amplified via the aRNA MessageAmp II kit (Ambion, 1751)
|
| Label |
Cy3
|
| Label protocol |
For each hybridization, 300 ng of Cy3 probes and 300 ng of Cy5 probes of amplified cRNA were mixed and added to 5 ul of 10x Blocking Agent, 1 ul of 25x Fragmentation Buffer and Nuclease free water in a 25 ul reaction, incubated at 60ºC for 30 minutes to fragment RNA and stopped with 25 ul of 2x Hybridization Buffer.
|
| |
|
| |
| Hybridization protocol |
The samples were loaded onto arrays, hybridized at 65ºC for 17 hours in a Hybridization oven rotator and then washed in GE wash buffer 1 at room temperature (1 minute) and in GE Wash Buffer 2 at 37ºC (1 minute). Arrays were dried by centrifugation at 2000 rpm for 2 minutes.
|
| Scan protocol |
Images from Cy3 and Cy5 channels were equilibrated and captured with a GenePix 4000B (Axon) and spots were converted into numerical data using GenPix software (Axon).
|
| Data processing |
Data processing was carried-out using the LIMMA package (Smyth, 2003) of Bioconductor project: Raw intensities were background-substracted by NORMEXP method with a offset of 50. Signals (in log2 scale) were then normalized by LOWESS algorithm (intra-arrays normalization) followed by adjustment of their quantiles (inter-arrays normalization). Linear model methods were then applied for determining differentially expressed genes. Each probe was tested for changes in expression over replicates by using an empirical Bayes moderated t-statistic.
|
| |
|
| Submission date |
Aug 01, 2016 |
| Last update date |
Mar 16, 2017 |
| Contact name |
Carlos Martinez |
| Organization name |
Centro Nacional de Biotecnologia
|
| Department |
Inmunology and Oncology
|
| Lab |
411
|
| Street address |
C/Darwin 3
|
| City |
Madrid |
| State/province |
Madrid |
| ZIP/Postal code |
28043 |
| Country |
Spain |
| |
|
| Platform ID |
GPL10333 |
| Series (2) |
| GSE85027 |
Dido as a switchboard that regulates self-renewal and differentiation in embryonic stem cells (vsmutNT) |
| GSE85029 |
Dido as a switchboard that regulates self-renewal and differentiation in embryonic stem cells |
|
