cell type: Human umbilical vein endothelial cells (HUVEC)
Growth protocol
Human umbilical vein endothelial cells (HUVEC) were cultured in gelatine coated 6-well plates with ECGM2 medium including Supplement Mix (PromoCell) and 10% FCS. Cells were grown as a confluent monolayer and transduced with adenovirus with an MOI of 50. 36 h later RNA was harvested.
Extracted molecule
total RNA
Extraction protocol
RNA was isolated using the Rneasy kit (Qiagen) according to the instructions of the manufacturer. Optional steps of the protocol were skipped and no optional supplements to the buffers were used. The quality of total RNA was checked by gel analysis using the total RNA Nano chip assay on an Agilent 2100 Bioanalyzer (Agilent Technologies). All samples had RNA index values greater than 8.5. RNA concentrations were determined using the NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE).
Label
biotin
Label protocol
Biotin-labeled cRNA were prepared according to Illumina's recommended sample labeling procedure based on the modified Eberwine protocol (Proc. Natl. Acad. Sci. U. S. A. (1992) 89:3010–3014). In brief, 250 ng total RNA was used for complementary DNA (cDNA) synthesis, followed by an amplification/labeling step (in vitro transcription) to synthesize biotin-labeled cRNA according to the MessageAmp II aRNA Amplification kit (Ambion, Inc., Austin, TX). Biotin-16-UTP was purchased from Roche Applied Science, Penzberg, Germany. The cRNA was column purified according to TotalPrep RNA Amplification Kit, and eluted in 60 µl of water. Quality of cRNA was controlled using the RNA Nano Chip Assay on an Agilent 2100 Bioanalyzer and spectrophotometrically quantified (NanoDrop).
Hybridization protocol
Hybridization is performed at 58°C, in GEX-HCB buffer (Illumina Inc.) at a concentration of 50 ng cRNA/µl, unsealed in a wet chamber for 20 h. Spike-in controls for low, medium and highly abundant RNAs were added, as well as mismatch control and biotinylation control oligonucleotides. Microarrays were washed twice in E1BC buffer (Illumina Inc.) at room temperature for 5 minutes. After blocking for 5 min in 4 ml of 1% (wt/vol) Blocker Casein in phosphate buffered saline Hammarsten grade (Pierce Biotechnology, Inc., Rockford, IL), array signals are developed by a 10-min incubation in 2 ml of 1 µg/ml Cy3-streptavidin (Amersham Biosciences, Buckinghamshire, UK) solution and 1% blocking solution. After a final wash in E1BC, the arrays are dried and scanned.
Scan protocol
Microarray scanning was done using a Beadstation array scanner, setting adjusted to a scaling factor of 1 and PMT settings at 430. Data extraction was done for all beads individually, and outliers are removed when > 2.5 MAD (median absolute deviation). All remaining data points are used for the calculation of the mean average signal for a given probe, and standard deviation for each probe was calculated.
Description
biological replicate 1
Data processing
The data were normalised using quantile normalisation with Illumina BeadStudio v. 3. For analysis duplicates were averaged and the expression fold change of Notch1 or ICAP1 expressing cells to the GFP expressing control was calculated
