cell type: Day 12 Embryoid Bodies differentiated from H9 hESCs developmental stage: Day 12
Growth protocol
hESCs were cultured on Matrigel-coated (ES qualified; BD Biosciences) tissue culture dishes with mTESR-1 human ES Growth Medium (StemCell Technologies). To induce endothelial differentiation, undifferentiated hESCs were cultured in differentiation medium containing Iscove’s modified Dulbecco’s medium (IMDM) and 15% Knockout TM Serum Replacement (KnockoutTM SR) (Invitrogen, Carlsbad, CA), 16 BIT (BSA, insulin, transferring; Stem Cell Technologies), 0.1 mM nonessential amino acids, 2 mM Lglutamine, 450 mM monothioglycerol (Sigma, St. Louis, MO), 50 U/ml penicillin, and 50 mg/ml streptomycin, supplemented with 20 ng/ml bFGF (R&D Systems Inc., Minneapolis) and 50 ng/ml VEGF (R&D Systems Inc.), in ultra-low attachment plates (Corning Incorporated, Corning, NY) for the formation of suspended embryoid bodies (EBs).12 days-old EBs were harvested, and then suspended into rat tail collagen type I (Becton Dickinson, San Jose, CA) at a final concentration of 1.5 mg/ml collagen. After thoroughly mixing EBs into collagen, 1.5 ml/well of mixture was added into six-well plate. The plates were incubated at 37C for 30 min, allowing gel polymerization prior to addition of medium. After gel formation, each dish was supplemented with EGM-2 medium (Lonza, Basel, Switzerland) and 5% KnockoutTM SR with additional 50 ng/ml VEGF and 20 ng/ml bFGF. The cultures were then incubated for 3 days without media change.
Extracted molecule
total RNA
Extraction protocol
Total RNA was prepared using RNeasy Mini Kit (Qiagen)
Label
Cy5
Label protocol
Using Low RNA Input Fluorescent Linear Amplification Kits (Agilent), cDNA was reverse transcribed from each RNA sample, as well as from a pooled reference control, and cRNA then transcribed and fluorescently labeled with Cy5/Cy3. cRNA was purified using an RNeasy kit (Qiagen).
cell type: pool of undifferentiated and differentiated hESCs, as well as fetal and neonatal tissues
Growth protocol
hESCs were cultured on Matrigel-coated (ES qualified; BD Biosciences) tissue culture dishes with mTESR-1 human ES Growth Medium (StemCell Technologies). To induce endothelial differentiation, undifferentiated hESCs were cultured in differentiation medium containing Iscove’s modified Dulbecco’s medium (IMDM) and 15% Knockout TM Serum Replacement (KnockoutTM SR) (Invitrogen, Carlsbad, CA), 16 BIT (BSA, insulin, transferring; Stem Cell Technologies), 0.1 mM nonessential amino acids, 2 mM Lglutamine, 450 mM monothioglycerol (Sigma, St. Louis, MO), 50 U/ml penicillin, and 50 mg/ml streptomycin, supplemented with 20 ng/ml bFGF (R&D Systems Inc., Minneapolis) and 50 ng/ml VEGF (R&D Systems Inc.), in ultra-low attachment plates (Corning Incorporated, Corning, NY) for the formation of suspended embryoid bodies (EBs).12 days-old EBs were harvested, and then suspended into rat tail collagen type I (Becton Dickinson, San Jose, CA) at a final concentration of 1.5 mg/ml collagen. After thoroughly mixing EBs into collagen, 1.5 ml/well of mixture was added into six-well plate. The plates were incubated at 37C for 30 min, allowing gel polymerization prior to addition of medium. After gel formation, each dish was supplemented with EGM-2 medium (Lonza, Basel, Switzerland) and 5% KnockoutTM SR with additional 50 ng/ml VEGF and 20 ng/ml bFGF. The cultures were then incubated for 3 days without media change.
Extracted molecule
total RNA
Extraction protocol
Total RNA was prepared using RNeasy Mini Kit (Qiagen)
Label
Cy3
Label protocol
Using Low RNA Input Fluorescent Linear Amplification Kits (Agilent), cDNA was reverse transcribed from each RNA sample, as well as from a pooled reference control, and cRNA then transcribed and fluorescently labeled with Cy5/Cy3. cRNA was purified using an RNeasy kit (Qiagen).
Hybridization protocol
825 ng of Cy3- and Cy5- labeled and amplified cRNA was mixed and fragmented according to the Agilent technology protocol. cRNA was hybridized to 4x44K whole human genome microarray slides from Agilent (Part G4112F) according to the manufacturer’s instructions. The hybridization was carried in a rotating hybridization chamber in the dark at 65°C for 17 h.
Scan protocol
The arrays were scanned using Agilent G2505B DNA microarray scanner.
Description
Day 12 Embryoid Bodies differentiated from H9 hESCs replicate 1
Data processing
The image files were extracted using Agilent Feature Extraction software version 9.5.1 applying LOWESS background subtraction and dye-normalization.