|
| Status |
Public on May 24, 2024 |
| Title |
Sirpα-/- glomeruli |
| Sample type |
RNA |
| |
|
| Source name |
glomeruli;Sirp-alpha-/-
|
| Organism |
Mus musculus |
| Characteristics |
gender: male
tissue: glomeruli
age: 8 month
|
| Treatment protocol |
The wild type (WT) and podocyte specific SIRPα knockout (Sirpa-/-) mice were received a single intraperitoneal injection of 0.5 ml pristane respectively, and monitored for 4 months to construct experimental LN mice. Podocytes were isolated from pristane-treated WT and Sirpa-/- mice for further analysis.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For the isolation of glomeruli, the renal cortex was cut into small cubes of ~1 mm3 and digested with HBSS solution containing collagenase D (1 mg/ml, Sigma-Aldrich, C6885), protease (1 mg/ml, Sigma-Aldrich, P6911) and DNase I (1 U/ml, Sigma-Aldrich, D5025) for 15 min at 37 °C. The digested tissue was screened through a 100 μm filter. Glomeruli containing Dynabeads were gathered using a magnetic particle concentrator. Adding 1ml Trizol (Vazyme, China) to each sample and extracting RNA according to the manufacturer's recommended protocol.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.25 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS 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-2000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 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 Scanner (G5761A) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression after in glomeruli from Sirpa-/- mice
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) 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 |
Aug 15, 2023 |
| Last update date |
May 24, 2024 |
| Contact name |
QIAN BIN |
| E-mail(s) |
[email protected]
|
| Phone |
15851836627
|
| Organization name |
State Key Laboratory of Natural Medicines
|
| Department |
School of Life Science and Technology
|
| Lab |
Jiangsu Key Laboratory of Druggability of Biopharmaceuticals
|
| Street address |
639 Longmian Avenue
|
| City |
Nanjing |
| State/province |
JiangSu |
| ZIP/Postal code |
211198 |
| Country |
China |
| |
|
| Platform ID |
GPL11202 |
| Series (1) |
| GSE240916 |
Loss of SIRPα promotes podocytes presenting antigen to activate specific T cell immune responses in lupus nephritis via activating spleen tyrosine kinase |
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