|
| Status |
Public on Feb 25, 2017 |
| Title |
Wt MEF Nuclear cDNA |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Wt MEF Nuclear cDNA
|
| Organism |
Mus musculus |
| Characteristics |
cell type: MEF
genotype: wt
|
| Growth protocol |
MEF cells were cultured in DMEM supplemented with 10% FCS, 50µg/ml Gentamicin, 2mM L-Glutamin, in 5% CO2 atmosphere at 37°C. Cells were split every 3rd day by washing with DPBS and trypsinizing cells with 0.25% Trypsin-EDTA.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from different tissues or cells. After the treatment of DNase I, total RNA was reverse transcribed, and synthesized to double-stranded cDNA.
|
| Label |
Cy5
|
| Label protocol |
1 µg ds-cDNA was directly labeled by Klenow (New England Biolabs) random priming with Cy3 nonamers per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
|
| |
|
| Channel 2 |
| Source name |
Wt MEF Cytoplasmic cDNA
|
| Organism |
Mus musculus |
| Characteristics |
cell type: MEF
genotype: wt
|
| Growth protocol |
MEF cells were cultured in DMEM supplemented with 10% FCS, 50µg/ml Gentamicin, 2mM L-Glutamin, in 5% CO2 atmosphere at 37°C. Cells were split every 3rd day by washing with DPBS and trypsinizing cells with 0.25% Trypsin-EDTA.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from different tissues or cells. After the treatment of DNase I, total RNA was reverse transcribed, and synthesized to double-stranded cDNA.
|
| Label |
Cy3
|
| Label protocol |
1 µg ds-cDNA was directly labeled by Klenow (New England Biolabs) random priming with Cy3 nonamers per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
|
| |
|
| |
| Hybridization protocol |
The labeled ds-cDNA was precipitated with 0.1 volume 5M NaCl and 1 volume isopropanol, and hybridized in 45 ul of buffer containing 20% formamide, 1.2 M betaine, 0.1 ug/ul herring sperm DNA and 10 ug of human COT1 DNA (Invitrogen). Arrays were hybridized in Maui hybridization stations for 16-18 h at 42C, and then washed in 42C 0.2% SDS/0.2x SSC, room temperature 0.2x SSC, and 0.05x SSC. Hybridization buffers and washes were completed using manufacturer's protocols (http://www.nimblegen.com/products/lit/lit.html).
|
| Scan protocol |
Arrays were scanned on an Axon 4000B scanner per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
|
| Description |
ch1 molecule: nuclear cDNA
ch2 molecule: cytoplasmic cDNA
Wt MEF Nuclear cDNA
|
| Data processing |
Arrays were processed using Nimblegen's standard protocol for Nimblescan 2.4 ChIP data extraction.
|
| |
|
| Submission date |
Jan 19, 2011 |
| Last update date |
Feb 25, 2017 |
| Contact name |
Florian M Pauler |
| E-mail(s) |
florian.pauler@ist.ac.at
|
| Phone |
+43 2243 9000-7434
|
| Organization name |
IST Austria
|
| Lab |
Simon Hippenmeyer
|
| Street address |
Am Campus 1
|
| City |
Klosterneuburg |
| ZIP/Postal code |
3400 |
| Country |
Austria |
| |
|
| Platform ID |
GPL11618 |
| Series (2) |
| GSE26718 |
Macro ncRNAs are abundant in imprinted regions and directly regulated by DNA methylation [tiling array] |
| GSE75454 |
Transcript identification and quantification on Mouse Imprinted Region Tiling Array (MIRTA) |
|
