|
| Status |
Public on Aug 02, 2012 |
| Title |
Sox9egfpneg_p23-2 |
| Sample type |
RNA |
| |
|
| Source name |
GFP- cells from Sox9-EGFP Mus Musculus pancreas at p23
|
| Organism |
Mus musculus |
| Characteristics |
background strain: CD1
age: p23
tissue: pancreas
genotype/variation: Sox9-EGFP
facs: GFP-
|
| Treatment protocol |
Pancreata from e10.5, e15.5, or p23 pancreata were dissected and dissociated with collagenase B and trypsin, if necessary, to create single cell suspensions that were subjected to FACS. Cells expressing high levels of GFP were collected for analysis for each population. For the Sox9-EGFPneg_p23 samples, the GFP negative cell population was also collected. Multiple animals were pooled before sorting for each population to achieve sufficient cell numbers.
|
| Growth protocol |
All animal experiments described herein were approved by the University of California, Irvine and San Diego Institutional Animal Care and Use Committees. Sox9-EGFP or Ngn3-EGFP males were crossed with CD1 (Charles River) wild-type females and embryos were harvested at e10.5 or e15.5. Alternatively, pancreata were harvested from Sox9-EGFP pups at p23. For timed matings, noon on the day of vaginal plug appearance was designated e0.5.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA isolation was performed with the Qiagen Micro RNA kit according to the manufacturer's instructions. The optional on column DNAse digestion was performed. Using the NuGEN Ovation RNA Amplification System V2 (NuGEN Technologies, San Carlos, CA) first strand cDNA was synthesized from the poly(A)+ mRNA present within the isolated total RNA (20 ng total RNA starting material used in each sample reaction). Fragmentation of the mRNA within the resulting cDNA/mRNA complexes created priming sites for DNA polymerase to synthesize DNA complementary to the first strand cDNAs. The resulting double stranded (ds) cDNAs have a unique DNA/RNA heteroduplex at one end. These ds DNA templates were used to perform linear isothermal DNA amplification (SPIA, NuGEN Technologies, San Carlos, CA).
|
| Label |
Biotin
|
| Label protocol |
Biotinylated cDNA were prepared according to the NuGEN FL-Ovation cDNA Biotin Module V2 protocol after fragmentation of 3.75 ug of the amplified cDNA (NuGEN Technologies, San Carlos CA).
|
| |
|
| Hybridization protocol |
After labeling the resulting fragmented, biotin-tagged cDNA (2.2 μg) was placed into a hybridization cocktail (220 μL), with 200 μL actually hybridized at 45°C with rotation for 18 hours (Affymetrix GeneChip Hybridization Oven 640) to probe sets present on an Affymetrix Mouse Gene 1.0 ST array. The GeneChip arrays were washed and then stained (SAPE, streptavidin-phycoerythrin) on an Affymetrix Fluidics Station 450 using the FS450_0004 fluidics protocol.
|
| Scan protocol |
Gene chips were scanned on an Affymetrix GeneChip 3000 Scanner 7G.
|
| Description |
Gene expression data from pancreatic Sox9- cells at p23
|
| Data processing |
The results were quantified and analyzed using Expression Console ver.1.1 software (Affymetrix, Inc.) using the PLIER Algorithm default values (Quantification Scale: Linear; Quantification Type: Signal and Detection P-Value; Background: PM-GCBG; Normalization Method: Sketch-Quantile).
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| |
|
| Submission date |
Nov 30, 2011 |
| Last update date |
Aug 02, 2012 |
| Contact name |
Brandon Taylor |
| Organization name |
University of California, San Diego
|
| Department |
Pediatrics and Cellular & Molecular Medicine
|
| Lab |
Maike Sander, M.D.
|
| Street address |
Sanford Consortium for Regenerative Medicine, 2880 Torrey Pines Scenic Dr., Room 3101
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
| |
|
| Platform ID |
GPL6246 |
| Series (1) |
| GSE34060 |
Expression data of Sox9+ and Ngn3+ mouse pancreas cells at different stages of development |
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