|
| Status |
Public on Dec 05, 2018 |
| Title |
nonTX,CD25- |
| Sample type |
RNA |
| |
|
| Source name |
no thymectomy, CD25-
|
| Organism |
Mus musculus |
| Characteristics |
tissue: spleen
cell type: T cells
cell markers: CD4+, CD25-
treatment: no thymectomy
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from mouse ear and purified using the RNeasy Mini Kit (Qiagen). Samples are pooled from 4 mice for each treatment. The quality of RNA was assessed with a 2100 Bioanalyzer (Agilent Technologies).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 150 ng RNA using the One-Color Low Input 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 |
1650 ng of Cy3-labelled cRNA (specific activity >6 pmol Cy3/µg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 µl containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint GE Unrestricted Microarrays (G2519F) 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.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 8x60K array slides (Scan Area 61x21.6 mm, Scan resolution 3 µm, Dye channel is set to Green and Green PMT is set to 100%).
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Dec 04, 2018 |
| Last update date |
Dec 05, 2018 |
| Contact name |
Naozumi Ishimaru |
| E-mail(s) |
ishimaru.n@tokushima-u.ac.jp
|
| Phone |
0886337328
|
| Organization name |
Tokushima University Graduate School
|
| Department |
Oral Molecular Pathology
|
| Street address |
Kuramoto
|
| City |
Tokushima |
| ZIP/Postal code |
7708504 |
| Country |
Japan |
| |
|
| Platform ID |
GPL11202 |
| Series (1) |
| GSE123332 |
Follicular helper T cells enhance autoimmunity through Ascl-2 in a murine model for Sjögren’s syndrome |
|
