|
| Status |
Public on Sep 30, 2011 |
| Title |
eGFP+ cells, biological rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Hb-eGFP-positive cells of electroporated chick embryos at stage HH5-6
|
| Organism |
Gallus gallus |
| Characteristics |
cell type: Hb-eGFP-positive cells
age: embryo
developmental stage: HH5-6
|
| Treatment protocol |
Embryos were microinjected and electroporated at stage HH3 with the vector Hb-eGFP, that contains a -255 to -204 bp fragment from the chick Cerberus regulatory region, and the human beta-globin minimal promoter (p1229-eGFP enhancerless vector).
|
| Growth protocol |
Chick embryos were incubated at 37.5ºC for approximately 12 hours
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Embryos were harvested at stage HH5-6 and dissociated into single cell suspensions. The eGFP-positive and eGFP-negative/RFP-positive cell populations were sorted by FACS using a Moflo high-speed cell sorter (Beckman Coulter). Cell populations were collected simultaneously into two different tubes, containing RNAlater (Ambion), and total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
RNA was processed for use on Affymetrix (Santa Clara, CA, USA) GeneChip Chicken Genome Arrays, according to the manufacturer’s Two-Cycle Target Labeling Assay. Briefly, 40 ng of total RNA containing spiked in Poly-A RNA controls (GeneChip Expression GeneChip Eukaryotic Poly-A RNA Control Kit; Affymetrix) was used in a reverse transcription reaction (Two-Cycle DNA synthesis kit; Affymetrix) to generate first-strand cDNA. After second-strand synthesis, double-stranded cDNA was used in an in vitro transcription (IVT) reaction to generate cRNA (MEGAscript T7 kit; Ambion, Austin, TX). 600 ng of the cRNA obtained was used for a second round of cDNA and cRNA synthesis, resulting in biotinylated cRNA (GeneChip Expression 3’-Amplification Reagents for IVT-Labeling; Affymetrix). Size distribution of the cRNA and fragmented cRNA, respectively, was assessed using an Agilent 2100 Bioanalyzer with a RNA 6000 Nano Assay.
|
| |
|
| Hybridization protocol |
15 µg of fragmented cRNA was used in a 300-µl hybridization containing added hybridization controls. 200 µl of mixture was hybridized on arrays for 16 h at 45°C. Standard post hybridization wash and double-stain protocols (FS450_0001) were used on an Affymetrix GeneChip Fluidics Station 450.
|
| Scan protocol |
Arrays were scanned on an Affymetrix GeneChip scanner 3000 7G.
|
| Description |
gene expression data of Hb-eGFP-positive cells
|
| Data processing |
Scanned arrays were analyzed first with GCOS 1.4 software to obtain Absent/Present calls and for subsequent analysis with dChip 2008. The arrays were normalized to a baseline array with median CEL intensity by applying an Invariant Set Normalization Method (Li and Wong, 2001). Normalized CEL intensities of the six arrays were used to obtain model-based gene expression indices based on a PM (Perfect Match)-only model.
|
| |
|
| Submission date |
Sep 29, 2011 |
| Last update date |
Sep 30, 2011 |
| Contact name |
Ana Teresa Tavares |
| E-mail(s) |
[email protected]
|
| Organization name |
IGC
|
| Street address |
Rua da Quinta Grande, 6
|
| City |
Oeiras |
| ZIP/Postal code |
2781-901 |
| Country |
Portugal |
| |
|
| Platform ID |
GPL3213 |
| Series (1) |
| GSE32494 |
Targeting the hemangioblast with a novel cell type-specific enhancer |
|
