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Status |
Public on Feb 25, 2017 |
Title |
CCE Day5 (Total cDNA, 1st biological replicate) |
Sample type |
mixed |
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Channel 1 |
Source name |
CCE Day5 (Total cDNA, 1st biological replicate)
|
Organism |
Mus musculus |
Characteristics |
cell type: Embryonic Stem Cell (ESC) time: day 5 genotype: CCE
|
Treatment protocol |
The differentiation of ESC by retinoic acid was induced by withdrawal of LIF and addition of 0.266µM retinoic acid (RA). Cells were plated at appropriate densities to be confluent at the day of harvesting. Cells were fed each third day and harvested at the indicated time points.
|
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 |
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.
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Channel 2 |
Source name |
Input (CCE Day5, sonicated genomic DNA)
|
Organism |
Mus musculus |
Characteristics |
cell type: Embryonic Stem Cell (ESC) time: day 5 genotype: CCE
|
Treatment protocol |
The differentiation of ESC by retinoic acid was induced by withdrawal of LIF and addition of 0.266µM retinoic acid (RA) to the medium (80ng/ml). Cells were plated at appropriate densities to be confluent at the day of harvesting. Cells were fed each third day and harvested at the indicated time points.
|
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 |
CCE Day5 (Total cDNA, 1st biological replicate)
|
Data processing |
Arrays were processed using Nimblegen's standard protocol for Nimblescan 2.4 ChIP data extraction.
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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) |
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