|
| Status |
Public on Dec 19, 2018 |
| Title |
Mouse 4- 2D- male |
| Sample type |
RNA |
| |
|
| Source name |
2D tissue culture of tibia and femur bone marrow stromal cells after 4 weeks of adipogenic differentiation
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
gender: male
sample type: 2D bone marrow cell
|
| Treatment protocol |
See growth protocol.
|
| Growth protocol |
Cells were cultured in basal growth media (DMEM supplemented with 10% FBS and 1% antibiotic-antimycotic) for 12 days and then switched to an adipogenic differentiation media (basal supplemented with 0.5 mM IBMX, 1 uM Rosi, 1 uM Dex, 10 ug/mL insulin) for 4 days, and then a seocnd adipogenic media (basal supplemented with 1 uM Rosi and 10 ug/mL insulin) for 3 days and then maintained in a third adipogenic media (basal supplemented with 10 ug/mL insulin only).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After 4 weeks of adipogenesis, total RNA was isolated from 10 samples using the Qiazol and miRNeasy isolation mini-kit (Qiagen) according to the manufacturer’s protocol. 100ng of RNA was used to synthesize cDNA through a first-strand reverse transcription reaction and second-strand RNA degradation reaction, using reagents from the GeneChip® WT PLUS Reagent Kit. cRNA was then synthesized through an overnight (16-hour) In-vitro transcription reaction, which utilizes a T7 RNA polymerase. The cRNA was purified using an Affymetrix® magnetic bead protocol. Sample concentrations were determined using a 40ug/mL/A260 constant on a Nanodrop 1000 spectrophotometer.
|
| Label |
Biotin
|
| Label protocol |
Approximately 5.5ug of single-stranded cDNA was fragmented using UDG (10U/uL) and APE1 (1000U/uL), provided in the GeneChip® WT PLUS Reagent Kit. Samples were then labeled with biotin using TdT (30U/uL), also provided in the GeneChip® WT PLUS Reagent Kit. Efficiency of the fragmentation and labeling reactions were verified using NeutrAvidin (10mg/mL) with a gel-shift assay.
|
| |
|
| Hybridization protocol |
Samples were combined with a hybridization mix, injected into Mouse ClariomTM S arrays, and place in the Affymetrix® GeneChip® Hybridization Oven 645 at 45° C and 60 RPM for 16.5 hours overnight.
|
| Scan protocol |
Arrays were stained using the Affymetrix® GeneChip® Fluidics Station 450 and scanned with the 7G Affymetrix® GeneChip® Scanner 3000. Raw image analysis was performed with Affymetrix® Expression ConsoleTM software. The raw data images produced from the scanner were processed into .CEL files, which contain measure intensities for each probe on the array.
|
| Description |
This is the fourth of five biological replicates of 2D bone marrow cell cultures from 16 week-old, C57BL/6J mice tibia and femurs.
|
| Data processing |
The .CEL files were imported into Partek® Genomics SuiteTM (GS) version 6.6 (Partek Inc., St. Louis, MO, USA, www.partek.com). Background correction, quantile normalization, log2 transformation, and probe set summarization were performed using default settings for the Robust Multichip Average (RMA) procedure (see Bolstad et al. Bioinformatics 19(2):185; Irizarry et al. Biostatistics 4(2):249; Nucleic Acids Res. 31:e15). A two-way Analysis of Variance (ANOVA) with factors matrix type and subject (paired design) was performed on the RMA-normalized probesets to assess differential expression.
|
| |
|
| Submission date |
Dec 20, 2017 |
| Last update date |
Dec 19, 2018 |
| Contact name |
Heather Elizabeth Driscoll |
| Organization name |
Norwich University
|
| Department |
Biology
|
| Street address |
158 Harmon Drive
|
| City |
Northfield |
| State/province |
VT |
| ZIP/Postal code |
05663 |
| Country |
USA |
| |
|
| Platform ID |
GPL23038 |
| Series (1) |
| GSE108374 |
Development of a 3D Bone Marrow Adipose Tissue Model |
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