|
| Status |
Public on Feb 25, 2017 |
| Title |
ThpM5 (Total cDNA, Thp/+ MEF) |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
ThpM5 (Total cDNA, Thp/+ MEF)
|
| Organism |
Mus musculus |
| Characteristics |
cell type: ThpM5
genotype: Thp/+
|
| 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 |
Labelling was performed by imaGenes GmbH (Berlin Germany). In brief: 1 µg ds-cDNA was directly labeled by Klenow (New England Biolabs) random priming with Cy5 nonamers.
|
| |
|
| Channel 2 |
| Source name |
Input (CCE Day0, sonicated genomic DNA)
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Embryonic Stem Cell (ESC)
time: day 0
genotype: CCE
|
| Growth protocol |
ESCs were cultured in Hepes-buffered DMEM, supplemented with Leukaemia Inhibitory Factor (LIF), 15% FCS, 50µg/ml Gentamicin, 1× MEM, 1mM NaPyruvate, 2mM L-Glutamin and 2mM β-mercaptoethanol, in 5% CO2 atmosphere at 37°C. For feeder independent ES cell culture, all dishes and flasks were gelatinized with 0.2% Gelatin solution (diluted in DPBS) at RT for 20min before use.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The genomic DNA isolated from CCE ES cell line was fragmented and used as Input DNA.
|
| Label |
Cy3
|
| Label protocol |
Labelling was performed by imaGenes GmbH (Berlin Germany). In brief: 1 µg ds-cDNA was directly labeled by Klenow (New England Biolabs) random priming with Cy3 nonamers.
|
| |
|
| |
| Hybridization protocol |
Hybridisation was performed by imaGenes GmbH (Berlin Germany). In brief: 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.
|
| Scan protocol |
Arrays were scanned on an Axon 4000B scanner per manufacturer's protocol by imaGenes GmbH (Berlin Germany).
|
| Description |
ThpM5 (Total cDNA, Thp/+ MEF)
|
| 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) |
|
