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Status |
Public on Aug 25, 2008 |
Title |
R-ES-NSC-5 |
Sample type |
RNA |
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Source name |
ES_differentiated neural stem cells
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Organism |
Homo sapiens |
Characteristics |
at pluripotent stage: NO Stem Cell Matrix: core dataset SCM core sNMF cluster: 9
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Growth protocol |
in vitro preparations were harvested, derived, differentiated and/or grown according to published protocols for the respective lines and samples. For further details and references please refer to Supplementary Tables 1 - 8 of the manuscript Regulatory networks define phenotypic classes of human stem cell lines
|
Extracted molecule |
total RNA |
Extraction protocol |
Depending on the lab that suplied samples, different extraction methods were used according to standard/manufacturers protocols: TriZOL extraction, Quiagen Rneasy Mini Kit or Ambion Mirvana
|
Label |
biotin
|
Label protocol |
total RNA samples were quality assessed using Nanodrop Spectrophotometer, Invitrogen Qbit Spectrophotometer and the Agilent Bioanalyzer. Samples were then amplified one round, using an RNA Amplification Kit (Ambion) according to the manufacturer's instructions. The resulting purified cDNA product of was resuspended and dried down in a speedvac centifuge. cDNA was then converted to cRNA using an in vitro transcription kit according to the manufacturer's instructions (Roche),followed by another round of QC with Nanodrop/Bioanalyzer
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Hybridization protocol |
Sucessfully labeled cRNA samples were then hybridized to Human Ref-8 BeadChips and Human WG6 BeadChips (Illumina) according to the manufacturer's instructions (approximately 23,000 identical gene probes are represented on both array designs), using equipment specified by the manufacturer (Illumina). Briefly, 850 ng biotin-labeled cRNA in 11.3 µl nuclease-free water was adjusted to 34 µl through the addition of 22.7 µl of 5:3 HybE1 buffer/formamide. The sample was heated at 65°C for 5 min, allowed to cool to room temperature, and then immediately added to a single array of an 8-array Human Ref-8 BeadChip. Once all 8 samples were added to each BeadChip, it was sealed in a Hyb Cartridge and incubated for 16 h at 55°C with rotation in an Illumina hybridization oven (rotation setting 5). Following overnight hybridization, BeadChips were moved to a slide rack and serially washed using gentle rotation in glass staining dishes filled with a) 250 ml Illumina Wash Buffer×5 min, b) 250 ml 100% ethanol×10 min, c) 250 ml Illumina Wash Buffer×2 min. BeadChips were then blocked for 10 min in 4 ml Block E1 buffer (Illumina), followed by staining for 10 min in 1 µg/ml Streptavidin-Cy3 conjugate (GE Healthcare) in Block E1 buffer. Stained BeadChips were finally washed using gentle rotation in a glass staining dish filled with 250 ml Illumina Wash Buffer×5 min. BeadChips were dried by centrifugation at 280×g for 4 min and stored in a light-tight box until reading.
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Scan protocol |
Processed arrays were read using a BeadStation array reader (Illumina) according to the manufacturer's instructions. Scan settings were for single color (green) scanning.
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Description |
1400364008E ES cells_ differentiated by shin_unpublished method
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Data processing |
All arrays (Human Ref-8 BeadChips and Human WG6 BeadChips) were uploaded in BeadStudio version 1.5.1.3 and the intensity values for all 23,000 identical probes on both array designs were extracted; Normalization = none; Array Content = Human_RefSeq-8.xml; Error Model = none; uploaded into R/Bioconductor; Quantile normalized with R/package Affy
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Submission date |
May 20, 2008 |
Last update date |
Aug 25, 2008 |
Contact name |
Franz-Josef Mueller |
E-mail(s) |
fj.mueller@zip-kiel.de
|
Phone |
0431-8006272
|
Fax |
0431-8006272
|
Organization name |
Zentrum für Integrative Psychiatrie
|
Lab |
Loring Lab Germany
|
Street address |
Niemannsweg 147
|
City |
Kiel |
State/province |
Schleswig-Holstein |
ZIP/Postal code |
24105 |
Country |
Germany |
|
|
Platform ID |
GPL2700 |
Series (1) |
GSE11508 |
Regulatory networks define phenotypic classes of human stem cell lines |
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