|
| Status |
Public on Mar 07, 2011 |
| Title |
AM-iPS-3 (Sage)_p7_1 |
| Sample type |
RNA |
| |
|
| Source name |
AM-iPS-3 (Sage), passage 7
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: AM-iPS-3 (Sage), passage 7
provenance somatic cell: human amniotic ectoderm and mesoderm layer (hAM) cell
iPS cell-line name: AM-iPS-3 (Sage)
passage number: 7
|
| Treatment protocol |
Human iPS cells were maintained in an undifferentiated state in the presence of bFGF. The cultures were incubated at 37 ºC in a humidified incubator with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using ISOGEN (Nippon Gene, Toyama, Japan) following the manufacturer's recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 1.0 ug RNA using the Quick-Amp Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.65 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ul of 2x HiRPM Hybridization Buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112F) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green PMT is set to 100%).
|
| Description |
Gene expression in human iPS cell
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.5.3 (Agilent) using default parameters (protocol GE1-v5_95 and Grid: 014850_D_20070820) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
Mar 11, 2010 |
| Last update date |
Nov 09, 2012 |
| Contact name |
Makoto Asashima |
| E-mail(s) |
[email protected]
|
| Phone |
+81-29-861-2529
|
| Fax |
+81-29-861-2987
|
| Organization name |
National Institute of Advanced Industrial Science Technology (AIST)
|
| Department |
Tsukuba Central 4
|
| Lab |
Research Center for Stem Cell Engineering
|
| Street address |
1-1-1 Higashi
|
| City |
Tsukuba |
| State/province |
Ibaraki |
| ZIP/Postal code |
305-8562 |
| Country |
Japan |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE20750 |
Gene expression signatures for human iPS cell lines |
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