Cells were treated with BMP4 (4ng/ml) or forskolin (25uM) or both for 8 hours.
Growth protocol
E11.5 aorta-gonad-mesonephros region was microdissected, dissociated using DNase I, Collagenase IV and Hyaluronidase, then sorted for VE-cadherin+ cells. Cells were cultured in serum-free media containing 2% BSA / IMDM (vol/vol) supplemented with Insulin-Transferrin-Selenium (ITS-G; Life Technologies), 25 μg/ml ascorbic acid, 450 μM monothioglycerol, 2 mM penicillin/streptomycin/glutamate and of 20 ng/ml VEGF (R&D), 50 ng/ml IL3 (R&D), 50 ng/ml IL6 (R&D), 50 ng/ml SCF (R&D), 50 ng/ml FLT3L (R&D), 50 ng/ml TPO (R&D), 50 ng/ml IL6R (SBH Sciences)
Extracted molecule
total RNA
Extraction protocol
RNA was extracted with RNeasy Micro Kit (Qiagen)
Label
biotin
Label protocol
NuGen amplified, biotinylated cRNA were prepared according to the standard Affymetrix protocol from total RNA.
Hybridization protocol
Following fragmentation, cRNA were hybridized for 16 hr at 45C on GeneChip Mouse Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
Scan protocol
GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.
Description
BMP4_rep_2 7_021014.CEL
Data processing
The data were filtered using nsFilter from the genefilter package and Multi-array Average-summarized, quantile normalized using R (v.2.15)/Bioconductor v.2.11 (BiocInstaller 1.8.3). After nsFilter (backup, var.filter=FALSE, require.entrez=FALSE, require.symbol=FALSE, remove.dupEntrez=TRUE) removing duplicate Entrez entries, the reduced data set of 20,757 rows was obtained.
