|
| Status |
Public on May 21, 2016 |
| Title |
pool of embryos 4, RFP+ GFP+ sorted population |
| Sample type |
RNA |
| |
|
| Source name |
Chick neuroepithelial cells at HH10/11 + 24 hpe , that activate BRE-reporter
|
| Organism |
Gallus gallus |
| Characteristics |
cell type: embryonic neuroepithelial cells
breed: white Leghorn chicken
age: HH10/11 + 24hours post-electroporation
|
| Treatment protocol |
A solution of plasmid DNA diluted in H2O and 0.2% Fast Green (Sigma) was microinjected into the lumen of the embryos’ NT and electrodes were placed either side of the NT, at trunk levels. Electroporation was carried out in ovo using a Intracel Dual Pulse TSS-100 electroporator, delivering five 50-ms pulses of 20 V, after which embryos were allowed to develop before harvesting at the time points indicated.
|
| Growth protocol |
Fertilized eggs of white Leghorn chickens were windowed after a 36/40-h incubation at 38C at 70% humidity, at which time the embryos have developed to HH stages (St) 10/11 (Hamburger and Hamilton, 1951).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Nearly 30 electroporated embryos were pooled, and a single cell suspension was obtained. GFP and RFP fluorescent cells were sorted using a MoFlo flow cytometer (DakoCytomation, Fort Collins, Colorado, USA). At least 100,000 RFP+ cells and 20,000 GFP+ cells were obtained per pool, and total RNA was extracted from the resulting cell sorted populations, 90% of RFP+/GFP+ cells or 90% of RFP+/GFP- cells, using a standard Trizol (Promega) protocol.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
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| |
|
| Hybridization protocol |
The quality and quantity of purified RNA was verified by measuring with nanodrop, and subsequently the RNA was hybridized to microarrays and processed in accordance to the manufacturer’s instructions (Affymetrix). Two-cycle cDNA synthesis was performed on 35–50 ng of total RNA and hybridized to the GeneChip Chicken Genome Array (Affymetrix)
|
| Scan protocol |
GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.
|
| Description |
Gene expression data from chick neuroepithelial cells at HH10/11 + 24 hpe , that activate Bone Morphogenetic Protein- responsive element.
c.BRE4
|
| Data processing |
For statistical analysis, the data from four biological replicates of each experiment were averaged and microarray data was analyzed using Bioconductor software, and a false discovery rate (FDR) with a p value <0.05. The quality of the data was assessed, normalized with the “rma” algorithm and differentially expressed genes were selected. The results were filtered using thresholds of [log2FC]>0.5849 and a p value <0.05. The excel file on the series record (BREvsCTRL_array_processed_data.xlsx) contains the statistical analysis.
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| |
|
| Submission date |
May 20, 2016 |
| Last update date |
May 21, 2016 |
| Contact name |
Elisa Martí |
| Organization name |
IBMB-CSIC
|
| Street address |
Baldiri i Reixac, 15-21
|
| City |
barcelona |
| State/province |
Not Listed |
| ZIP/Postal code |
08028 |
| Country |
Spain |
| |
|
| Platform ID |
GPL3213 |
| Series (1) |
| GSE81717 |
Delamination of neural crest cells requires transient and reversible Wnt inhibition mediated by Dact1/2 |
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