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Sample GSM499743 Query DataSets for GSM499743
Status Public on Jan 28, 2010
Title EB-1
Sample type RNA
 
Channel 1
Source name EB
Organism Homo sapiens
Characteristics 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).
 
Channel 2
Source name Pooled RNA reference
Organism Homo sapiens
Characteristics 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.
 
Submission date Jan 22, 2010
Last update date Jan 27, 2010
Contact name Kitchener D. Wilson
E-mail(s) kitchwilson@stanford.edu
Organization name Stanford University
Street address S140 Grant Bldg
City Stanford
State/province CA
ZIP/Postal code 94305
Country USA
 
Platform ID GPL6480
Series (1)
GSE20013 Transcriptional Characterization of Human Embryonic Stem Cell-Derived Endothelial Cells

Data table header descriptions
ID_REF
VALUE normalized log10 ratio (Cy5/Cy3; test/ref)

Data table
ID_REF VALUE
A_24_P66027 -1.4351797
A_32_P77178 -0.048126146
A_23_P212522 0.83303535
A_24_P934473 -0.52926713
A_24_P9671 -0.10456654
A_32_P29551 0.28531656
A_24_P801451 -0.64798933
A_32_P30710 0.8852462
A_32_P89523 -0.7971613
A_24_P704878 -0.37918732
A_32_P86028 0.00191307
A_24_P470079 0.4275855
A_23_P65830 -0.2807332
A_23_P109143 -0.35442346
A_24_P595567 2.356386
A_24_P391591 1.7881079
A_24_P799245 0.9992224
A_24_P932757 0.88612777
A_24_P835500 0.26136297
A_23_P54340 -1.8478116

Total number of rows: 41076

Table truncated, full table size 946 Kbytes.




Supplementary file Size Download File type/resource
GSM499743.txt.gz 15.5 Mb (ftp)(http) TXT
Processed data included within Sample table

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