|
| Status |
Public on Jul 09, 2014 |
| Title |
Wildtype_CD34negative_keratinocytes |
| Sample type |
RNA |
| |
|
| Source name |
CD34 negative keratinocytes from Wild type mice
|
| Organism |
Mus musculus |
| Characteristics |
strain/background: CD1
genotype: wild type
gender: male+female
number: pool of 11 mice
age: 8 weeks
tissue: skin
cell type: CD34 negative keratinocytes
|
| Biomaterial provider |
Bram De Craene
|
| Treatment protocol |
Preparation of keratinocytes of 8-week old mice: the back skins of adult mice were incubated overnight on a 1X trypsin solution (Gibco BRL cat # 25050-014). Harvesting of keratinocytes was done the next day according to the procedure described in (1). CD34 positive cells were selected with a biotinylated CD34 antibody (eBioscience cat #13-0341-85) using the easySEP mouse biotin kit (Stem Cell Technologies cat #18556) and easySEP magnet (Stem Cell Technologies cat #18000) following the manufacturer’s instructions. Mts24 positive cells were selected using a specific antibody (2), followed by incubation with an anti-rat PE labelled secondary antibody (BD Biosciences #550767). Cells were then purified further using the easySEP mouse PE kit (Stem Cell Technologies cat #18554) and easySEP magnet, following the manufacturer’s instructions. References (1) Jensen KB, Driskell RR, Watt FM. Assaying proliferation and differentiation capacity of stem cells using disaggregated adult mouse epidermis. Nat Protoc 2010; 5 (5): 898-911., (2) Raymond K, Richter A, Kreft M, Frijns E, Janssen H, Slijper M, et al. Expression of the orphan protein Plet-1 during trichilemmal differentiation of anagen hair follicles. The Journal of investigative dermatology 2010 Jun; 130 (6): 1500-1513.
|
| Growth protocol |
Mice were housed and experiments were done in accordance with the institutional guidelines regarding the care and use of laboratory animals.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared with Trisure (Bioline cat #BIO-38032) following the manufacturer’s instructions
|
| Label |
biotin
|
| Label protocol |
RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyser 2100 (Agilent). Using the Ambion WT Expression Kit, per sample, an amount of 100 ng of total RNA spiked with bacterial poly-A RNA positive controls (Affymetrix) was converted to double stranded cDNA in a reverse transcription reaction. Next the sample was converted and amplified to antisense cRNA in an in vitro transcription reaction which was subsequently converted to single stranded sense cDNA. Finally, samples were fragmented and labeled with biotin in a terminal labeling reaction according to the Affymetrix WT Terminal Labeling Kit.
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| |
|
| Hybridization protocol |
A mixture of fragmented biotinylated cDNA and hybridisation controls (Affymetrix) was hybridised on Affymetrix Mouse Gene 1.0 ST Arrays followed by staining and washing in a GeneChip® fluidics station 450 (Affymetrix) according to the manufacturer’s procedures.
|
| Scan protocol |
To assess the raw probe signal intensities, chips were scanned using a GeneChip® scanner 3000 (Affymetrix).
|
| Description |
Sample preparation was done at VIB-UGent (Ghent, Belgium), from labeling on experiments were done at Nucleomics (Leuven, Belgium), result interpretation was done at VIB-UGent (Ghent, Belgium)
|
| Data processing |
Data were processed with RMA as implemented in the XPS package of Bioconductor. Reported values are on log2-scale.
|
| |
|
| Submission date |
Jul 15, 2013 |
| Last update date |
Jul 09, 2014 |
| Contact name |
Rekin's Janky |
| E-mail(s) |
[email protected]
|
| Organization name |
VIB
|
| Department |
Nucleomics Core
|
| Street address |
Herestraat 49 Box 816
|
| City |
Leuven |
| ZIP/Postal code |
B-3000 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL6246 |
| Series (1) |
| GSE48859 |
Study of stem cells and progenitor cells in K14 Snail mice |
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