BOEC were always harvested at passage 3, which comprised a nominal million-fold expansion since establishing culture and amounted to ~3x107 BOEC. They were always harvested 4 hours after the last change of culture medium, and always when they were at 85-90% confluent.
Growth protocol
We obtained citrated fresh peripheral blood (50-100 ml) from each donor. For donors outside the Minneapolis metropolitan area, blood was shipped by same-day express delivery in Saf-T-Pak cartons that had been pre-equilibrated to room temperature. Samples were processed immediately upon arrival at the University of Minnesota. Blood buffy coat mononuclear cells were prepared and cultured on rat collagen I, in presence of endothelial cell growth factors, as previous described in detail 15,16. All samples were handled identically
Extracted molecule
total RNA
Extraction protocol
We prepared BOEC lysates by adding 10 ml trizol (Gibco) to each T75 flask to solubilize the sample. As necessary, trizol lysates were stored in liquid nitrogen. We then isolated total RNA, and cleaned it using an RNeasy kit (Qiagen, CA).
Label
biotin
Label protocol
We used the Invitrogen SuperScript Choice system to reverse transcribe and synthesize double-stranded cDNA. For in vitro transcription and biotin-labeling, we synthesized biotin-labeled cRNA using the Enzo Life Sciences BioArray High Yield RNA Transcript Labeling kit. Then fragmentation of cRNA was done by standard protocol, and fragmentation was verified by gel electrophoresis.
Hybridization protocol
After we prepared biotin-labeled cRNA fragments, samples were turned over immediately to our Microarray Core Facility which used Gene Chip Hybridization Oven 640, Affymetrix FS4000 Fluidics Station for staining with a streptavidin-fluorochrome probe and washing,
Scan protocol
An Affymetrix High Resolution Scanner was used according to the manufacturer's standard manual
Description
BOEC (blood outgrowth endothelial cells) from 20 subjects with sickle cell anemia shown to be either at-risk or not-at-risk for ischemic stroke
Data processing
For raw microarray data, we used the RMA (robust multi-array average) method to summarize expression values from probe pair values; data were background-adjusted, quantile normalized (global scaling across multiple arrays).
Genetic Endothelial Systems Biology of Sickle Stroke Risk
Data table header descriptions
ID_REF
VALUE
Expression measures were summarized based on log transformed PM (perfect match) values using Median Polish algorithm. Then, we used locally weighted scatterplot smoothing (LOWESS) to do within-array normalization. RMA and LOWESS were performed in Genedata Expressionist Pro3.1® (Basel, Switzerland).
