|
| Status |
Public on Aug 15, 2018 |
| Title |
wildtype mice 3 |
| Sample type |
RNA |
| |
|
| Source name |
WT BMSCs replicate 3
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Bone marrow stem cells
gender: male
|
| Treatment protocol |
Femurs of mice were collected and crushed with a mortar and pestle. The bone was then digested with collagenase A (Sigma) to obtain a single-cell suspension. Cells within the supernatant were collected and incubated with phycoerythrin (PE)-, FITC-, peridinin chlorophyll protein (PerCP)- and allophycocyanin (APC)-conjugated antibodies that recognized mouse Sca-1 (Cat. No. 108108, BioLegend), CD29 (Cat. No. 102206, BioLegend), CD45 (Cat. No. 103132, BioLegend), and CD11b (Cat. No. 101226, BioLegend) for 20 min at 4°C. The acquisition was performed on a fluorescence-activated cell sorting (FACS) Aria model (BD Biosciences), and the analysis was performed using FACS DIVE software version 6.1.3 (BD Biosciences).
|
| Growth protocol |
The sorted mouse Sca-1+ CD29+ CD45− CD11b− BMSCs were cultured to reach 80%–85% confluence. Then, first-passage BMSCs were detached and seeded in culture flasks for the enrichment of cell populations.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the Versagene Blood RNA Purification kit (Gentra Systems, Minneapolis MN) following the manufacturer's recommendations. The protocol includes differential lysis of red and white blood cells, and an on-column DNase digestion. Globin message was further reduced using GLOBINclear (Ambion Inc., Austin, TX) to specifically remove both a- and b- globin. 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 0.5 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-1000 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 DNA Microarray Scanner (G2505B) 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 6hr in 2Gy-irradiated human blood
|
| 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.
|
| |
|
| Submission date |
Jul 11, 2018 |
| Last update date |
Aug 15, 2018 |
| Contact name |
CHANGJUN LI |
| E-mail(s) |
lichangjun@csu.edu.cn
|
| Organization name |
Xiangya Hospital of Central South University
|
| Department |
Department of Endocrinology, Endocrinology Research Center
|
| Street address |
87# Xiangya Road
|
| City |
changsha |
| ZIP/Postal code |
410008 |
| Country |
China |
| |
|
| Platform ID |
GPL21163 |
| Series (1) |
| GSE116926 |
Gene expression in BMSCs isolated from NONMMUT002667 gene KO mice and wildtype control mice |
|
