7 protocols
feature extraction protocol
The array images were registered using an algorithm described previously (Ritchie ME, Dunning MJ, Smith ML, Shi W, Lynch AG (2011) BeadArray Expression Analysis Using Bioconductor. PLoS Comput Biol 7(12): e1002276. doi:10.1371/journal.pcbi.1002276). Essentially the bead signals were computed with weighted averages of pixel intensities, and local background is subtracted. Sequence-type signal was calculated by averaging corresponding bead signals with outliers removed (using median absolute deviation). Preliminary data analysis and QC was carried out using the BeadStudio software (Illumina)
array scanning protocol
The arrays were scanned on the Illumina BeadArray Reader, a confocal-type imaging system with 532 (cye3) nm laser illumination
nucleic acid hybridization to array protocol
Hybridization of labeled cRNA to the BeadChip, and washing and scanning were performed according to the Illumina BeadStation 500x manual. Essentially the amplified, biotin-labeled human cRNA samples were resuspended in a solution of Hyb E1 buffer (Illumina) and 25% (v/v) formamide at a final concentration of 25 ng/uL. 1.5 ug of each cRNA were hybridized. Hybridization was allowed to proceed at 55 C, for 18 hours after which, the bead array matrix was washed for 10 minutes with 1X High temperature buffer (Illumina), followed by a subsequent 10 minute wash in Wash E1BC buffer. The arrays were then washed with 100% ethanol for 10 min to strip off any remaining adhesive on the chip. A 2 minute E1BC wash was performed to remove residual ethanol. The arrays were blocked for 5 minutes with 1% (w/v) casein-PBS, (Pierce). The array signal was developed via 10 minute incubation with Streptavidin-Cy3 at a final concentration of 1ug/mL solution of (GE Healthcare) in 1% casein-PBS blocking solution. The Mouse 6 Sentrix Expression BeadChip was washed a final time in Wash E1BC buffer for five minutes and subsequently dried via centrifugation for 4 minutes at a setting of 275 rcf
nucleic acid labeling protocol
Biotynilated cRNA was prepared using a modified Eberwine protocol ('t Hoen PA, de Kort F, van Ommen GJ, den Dunnen JT. Fluorescent labelling of cRNA for microarray applications.Nucleic Acids Res. 2003 Mar 1;31(5):e20.), by which messenger RNA is converted to cDNA, followed by an amplification/labeling step mediated by T7 DNA polymerase. The cDNA and cRNA filter cartridges (Ambion) were used according to the manufacturer's instructions for RT and IVT cleanup, respectively
nucleic acid extraction protocol
Total RNA was extracted from cells using an Rneasy kit from Qiagen Inc. (Valencia, CA).
treatment protocol
The hESC lines SA461 (Cellartis, Dundee, Scotland) were cultured in a feeder-free system. To maintain hESCs in prolonged pluripotent status, hESCs were cultured at 2_105 cells/ cm2 on fibronectin (Merck Chemicals Ltd, Nottingham, England). Cells were cultured in chemically defined media16 (denoted “pluripotent maintenance media”), supplemented with 50% human umbilical Wharton inner layer (Cellartis) fibroblast-conditioned media (VitrohES; VitroLife, Gothenburg, Sweden) and 10 ng/mL basic fibroblast growth factor (Invitrogen, Paisley, Scotland). Endothelial differentiation was induced by incubation in an “endothelial differentiation media,” consisting of large-vessel endothelial growth media, 500 mL (TCS CellWorks, Buckingham, England), supplemented with hydrocortisone, 1 ug/mL; human epidermal growth factor, 10 ng/mL; basic fibroblast growth factor, 3 ng/mL; and heparin, 10 ug/mL (TCS CellWorks).
growth protocol
The hESC lines SA461 was obtained from (Cellartis, Dundee, U.K.). Pluripotency was maintained and endothelial differentiation induced as previously described [1]. Primary human saphenous vein ECs (HSVECs) were isolated on the day of surgery by standard collagenase digestion based on a modified version of the protocol described by Jaffe and colleagues [2]. [1] Kane NM, Meloni M, Spencer HL, Craig MA, Strehl R, Milligan G, Houslay MD, Mountford JC, Emanueli C, Baker AH: Derivation of endothelial cells from human embryonic stem cells by directed differentiation: analysis of microRNA and angiogenesis in vitro and in vivo. Arterioscler Thromb Vasc Biol 2010, 30: 1389-1397. [2] Jaffe EA, Nachman RL, Becker CG, Minick CR: Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 1973, 52: 2745-2756.