|
| Status |
Public on May 01, 2014 |
| Title |
H9_HEPs_DLL4+ |
| Sample type |
RNA |
| |
|
| Source name |
H9-derived DLL4high HEPs
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: hESC-derived HEPs
cell line: H9
|
| Growth protocol |
Undifferentiated hESCs at confluence were treated with collagenase IV and scraped off of the Matrigel attachments. To allow human embryoid body (hEB) formation, hESC clumps were transferred to low-attachment plates (Corning) and incubated overnight in differentiation medium [DM; Knock-out-Dulbecco’s modified Eagle’s medium (KO-DMEM) supplemented with 20% non-heat-inactivated fetal bovine serum (FBS), 1% nonessential amino acids (NEAA), 1 mmol/L l-glutamine, and 0.1 mM β-mercaptoethanol]. The medium was changed the next day (day 1) with the same DM supplemented with hematopoietic cytokines: 300 ng/mL stem cell factor (SCF), 300 ng/mL Flt3L, 10 ng/mL interleukin (IL)-3, 10 ng/mL IL-6, 50 ng/mL granulocyte-colony stimulating factor (G-CSF) and 25 ng/mL bone morphogenetic protein 4 (BMP-4)19, 54. hEBs were dissociated using collagenase B (Roche Diagnostic) for 2 h at 37ºC followed by 10min incubation at 37ºC with enzyme-free Cell Dissociation Buffer (Invitrogen). A single-cell suspension was obtained by gentle pipetting and passage through a 70-µm cell strainer. The dissociated cells were stained with anti-CD34-PE-Cy7(BD Biosciences), anti-CD31-PE, anti-CD45-APC or -FITC and DLL4-APC or -PE antibodies (all from Miltenyi) and 7-actinomycinD (7-AAD, BD Biosciences). CD31+CD34+CD45-DLL4high and CD31+CD34+CD45-DLL4low/- HEPs were isolated using a FACSAria sorter (Becton Dickinson).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using standard RNA extraction protocols (NucleoSpin® RNA II). The RNA samples were quality-checked via the Agilent 2100 Bioanalyzer platform (Agilent Technologies)
|
| Label |
Cy3
|
| Label protocol |
For the linear T7-based amplification step, 70 ng of each total RNA sample was used. To produce Cy3-labeled cRNA, the RNA samples were amplified and labeled using the Agilent Low Input Quick Amp Labeling Kit (Agilent Technologies) following the manufacturer’s protocol. Yields of cRNA and the dye-incorporation rate were measured with the ND-1000 Spectrophotometer (NanoDrop Technologies).
|
| |
|
| Hybridization protocol |
The hybridization procedure was performed according to the Agilent 60-mer oligo microarray processing protocol using the Agilent Gene Expression Hybridization Kit (Agilent Technologies). Briefly, 600 ng Cy3-labeled fragmented cRNA in hybridization buffer was hybridized overnight (17 hours, 65 °C) to Agilent Whole Human Genome Oligo Microarrays 8x60K using Agilent’s recommended hybridization chamber and oven. Finally, the microarrays were washed once with the Agilent Gene Expression Wash Buffer 1 for 1 min at room temperature followed by a second wash with preheated Agilent Gene Expression Wash Buffer 2 (37 °C) for 1 min. The last washing step was performed with acetonitrile.
|
| Scan protocol |
Fluorescence signals of the hybridized Agilent Microarrays were detected using Agilent’s Microarray Scanner System (Agilent Technologies).
|
| Description |
Gene expression in HEPs with high surface expression of DLL4
|
| Data processing |
The Agilent Feature Extraction Software (FES) was used to read out and process the microarray image files. The software determines feature intensities (including background subtraction), rejects outliers and calculates statistical confidences. For determination of differential gene expression FES derived output data files were further analyzed using the Rosetta Resolverâ gene expression data analysis system (Rosetta Biosoftware).
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| |
|
| Submission date |
Apr 17, 2014 |
| Last update date |
May 01, 2014 |
| Contact name |
Veronica Ayllon |
| E-mail(s) |
[email protected]
|
| Organization name |
GENYO. Centre for Genomics and Oncological Research
|
| Street address |
Av Ilustracion, 114
|
| City |
Granada |
| ZIP/Postal code |
18016 |
| Country |
Spain |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE56881 |
Gene expression profile of DLL4+ and DLL4- Hemato-Endothelial Progenitors (HEPs) subpopulations |
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