|
| Status |
Public on Jun 03, 2010 |
| Title |
T cells |
| Sample type |
RNA |
| |
|
| Source name |
T cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: T cells
|
| Treatment protocol |
iPSCs and ESCs were collected by standard collagenase treatment. Activated T cells were collected by cell scraping.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the Trizol and RNeasy Kit following the manufacturer's recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.4 ug RNA using the One-Color 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 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) 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 (G2505CA) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to XDR Hi 100% and XDR lo 10%).
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.5 (Agilent) using default parameters (protocol GE1-105_Apr08 and Grid: 014850_D_20090416) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Jun 02, 2010 |
| Last update date |
Jun 02, 2010 |
| Contact name |
Shinsuke Yuasa |
| E-mail(s) |
yuasa@a8.keio.jp
|
| Organization name |
Keio University
|
| Street address |
35-Shinanomachi Shinjuku-ku
|
| City |
Tokyo |
| ZIP/Postal code |
160-8582 |
| Country |
Japan |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE22088 |
Gene expression profiles of TiPS, HDF-iPS, ES and T cells |
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