normalization data transformation protocol
Data were analyzed using the limma and affy Bioconductor packages implemented in the R-statistical environment. RMA was used to normalize the data and fit log2 transformed expression values. ID_REF = VALUE = Log 2 Transformed RMA
array scanning protocol
GeneChips were scanned using the Affymetrix 3000 7G GeneChip Scanner with Autoloader.
Following Labeling, 4 ug of cDNA were hybridized for 16 hr at 45C on a Mouse 430 2.0 cartridge array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
Biotinylated cDNA were prepared according to the Nugen WT-Pico Ovation kit protocol from 10 ng total RNA (WT-Pico Ovation RNA Amplification System Manual, 2007, NuGEN). Subsequently cDNA is made double stranded fragmented and end-labeled following manufacturers standard protocol (NuGEN exon module, encore end-labeling module).
Embryos were dissected and single cell suspensions were made from the intermediate mesoderm region. EGFP positive cells were isolated by FACS
nucleic acid extraction protocol
RNA was isolated from EGFP positive cells using the RNA easy mini kit from Qiagen