|
| Status |
Public on Dec 31, 2014 |
| Title |
VSMC SA001 day6 batch c |
| Sample type |
RNA |
| |
|
| Source name |
Human Embryonic Stem Cell line SA001 from Cellartis AB differentiated into Vascular Smooth Muscle Cells; batch c
|
| Organism |
Homo sapiens |
| Characteristics |
phenotype: normal-VSMC_day6
|
| Treatment protocol |
cells were flash frozen and stored at -80°C until RNA extraction
|
| Growth protocol |
Primary endothelial and vascular smooth muscle cells were cultured at 37°C, 5% CO2 and 95% humidity in EGM2 medium (Lonza) until they reached a confluency of maximal 80%.
The Human Embryonic Stem Cell line SA001 (Cellartis AB) was cultured in Priming Medium, consisting N2B27 medium (1:1 mixture of DMEM:F12 (1:1) with Glutamax (Invitrogen) and Neurobasal media supplemented with N2 and B27 (all Invitrogen)) with 1 μM CP21R7 (Roche) and 25 ng/ml BMP4 (R&D Systems)
For differentiation into VSMCs, Human Embryonic Stem Cell line SA001 was cultured in VSMC Induction Medium consisting of N2B27 medium supplemented with 10ng/ml PDGF-BB and 2ng/ml Activin
For differentiation into VSMCs, Human Embryonic Stem Cell line SA001 was cultured in VSMC Expansion Medium consisting of N2B27 with 2ug/ml Heparin and 2ng/ml ActivinA
For differentiation into ECs, Human Embryonic Stem Cell line SA001 was cultured in EC Induction Medium consisting of StemPro-34 SFM medium (Invitrogen) supplemented with 200ng/ml VEGF (PeproTech) and 2μM Forskolin (Sigma-Aldrich)
For differentiation into ECs, Human Embryonic Stem Cell line SA001 was cultured in EC Expansion Medium consisting of StemPro-34 SFM supplemented with 50 ng/ml VEGF
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the RNeasy Mini kit (Qiagen Inc., Valencia, CA, USA) . DNA was removed by on-column DNase digestion with the RNase-Free DNase set (Qiagen). RNA quality and concentration was determined by analysis with an Agilent 2100 bioanalyzer (Agilent Technologies Inc., Santa Clara, CA, USA).
|
| Label |
Cy3
|
| Label protocol |
Labeling was performed using 5 ug of total RNA input following the standard operating protocol of NimbleGen Systems Inc., Madison, WI USA. See www.nimblegen.com.
|
| |
|
| Hybridization protocol |
Hybridization was performed following the standard operating protocol of NimbleGen Systems Inc., Madison, WI USA. See www.nimblegen.com.
|
| Scan protocol |
Scanning was performed on a NimbleGen MS200 Microarray Scanner at 2 μm pixel resolution following the standard operating protocol of NimbleGen Systems Inc., Madison, WI USA. See www.nimblegen.com.
|
| Description |
VSMCs in vitro differentiated from HESC SA001 depleted with CD144 Microbeads and an autoMACSpro (Miltenyi Biotec) and cultivated in VSMC Induction Medium on gelatin coated wells. Total cultivation time starting from HESC plating is 6 days (day 6). It is the third of three wild-type replicates used in this experiment, each from separate cultures.
|
| Data processing |
The raw data (.pair file) was subjected to background correction, quantile normalization (Bolstad et al. Bioinformatics 19(2):185), and RMA (Robust Multi-Array Analysis; Irizarry et al. Biostatistics 4(2):249) as implemented in the NimbleScan software package, version 2.6 (Roche NimbleGen, Inc.).
|
| |
|
| Submission date |
Jul 11, 2014 |
| Last update date |
Dec 31, 2014 |
| Contact name |
Tobias Heckel |
| E-mail(s) |
tobias-heckel@t-online.de
|
| Organization name |
Roche
|
| Department |
pRED
|
| Lab |
Safety Genetics & Genomics
|
| Street address |
Grenzacherstr. 124
|
| City |
Basel |
| ZIP/Postal code |
CH-4070 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL16025 |
| Series (1) |
| GSE59326 |
Expression profiling in primary and stem-cell derived Human Endothelial and Vascular Smooth Muscle Cells |
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