|
| Status |
Public on Aug 09, 2013 |
| Title |
MEF_Wapl(+/F)_1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
G0 phase synchronized MEFs
|
| Organism |
Mus musculus |
| Characteristics |
strain: mixed C57BL/6 and 129
cell type: G0 phase synchronized MEFs
age: 13.5 dpc
sample group: B1
genotype: Wapl (+/F) ERCre/+
treatment: starvation
hybridisation chip: 58MM
|
| Treatment protocol |
4-OHT was added to the starvation medium to a final concentration of 500 nM
|
| Growth protocol |
MEFs were starved in OptMEM containing 2% serum for seven days before RNA extraction
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using RNeasy Mini Kit (QIAGEN) following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
Labeling with a Reactive Fluorescent Dye/ Molecular probes (Alexa 555, 647) Thoroughly dissolve the amine-modified 4µg DNA in 5µl nuclease free H2O (warming to 42°C for 5 min if necessary) Add 3µl Labeling buffer (Just before use dissolve one vial of reactive dye in 10-20µl DMSO or use aliquots from –80°C) Add 2µl reactive dye to the samples, vortex briefly to mix (do not spin) Incubate for 1 hour in the dark at room temperature
|
| |
|
| Channel 2 |
| Source name |
pool
|
| Organism |
Mus musculus |
| Characteristics |
reference: pool
|
| Treatment protocol |
4-OHT was added to the starvation medium to a final concentration of 500 nM
|
| Growth protocol |
MEFs were starved in OptMEM containing 2% serum for seven days before RNA extraction
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using RNeasy Mini Kit (QIAGEN) following manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
Labeling with a Reactive Fluorescent Dye/ Molecular probes (Alexa 555, 647) Thoroughly dissolve the amine-modified 4µg DNA in 5µl nuclease free H2O (warming to 42°C for 5 min if necessary) Add 3µl Labeling buffer (Just before use dissolve one vial of reactive dye in 10-20µl DMSO or use aliquots from –80°C) Add 2µl reactive dye to the samples, vortex briefly to mix (do not spin) Incubate for 1 hour in the dark at room temperature
|
| |
|
| |
| Hybridization protocol |
Probe- Prehybridisation (TECAN HS4800) Protocol for 40 domain chips: Combine: 50µl Labeled DNA 29µl 20x SSPE 4µl 50x Denhardts solution 50µl Form amid 1.5µl mouse block 3.5µl 20% SDS (last component to be added) Incubate at 94°C for 2 min Prehybridise probe for at least 1 hour at 50°C • start prehybridization of the slides • After prehyb of the samples and the slides pipette 135 µl onto each slide If you have duplicates then pool and use 135µl per slide • split and pipette 140 µl onto each slide • Continue with hybridization on TECAN HS4800 for 12hours 45°C
|
| Scan protocol |
Scanner: GenePix 4000B
|
| Description |
Wapl (+/F)
|
| Data processing |
Image Analysis: GenPix Pro 6.0. Data analysis: The statistical computing software R version 2.11.0 http://www.R-project.org and the limma package [Smyth 2004] was used for data normalization and calculation of fold changes and differential expression. In brief, we first removed all clones with missing signal intensities. Micro Array expression intensities were then background corrected using RMA and normalized within arrays with default settings. Only clones with reasonable (>250) expression values in at least 25% of the samples were analyzed further. Differentially expressed genes were obtained by applying limma's linear model fit and subsequent Benjamini Hochberg p-value adjustment. Genes with adjusted p-value smaller than 0.01 were called significant.
|
| |
|
| Submission date |
Oct 10, 2012 |
| Last update date |
Aug 09, 2013 |
| Contact name |
Roman R Stocsits |
| E-mail(s) |
roman.stocsits@imp.ac.at
|
| Organization name |
IMP - Research Institute of Molecular Pathology
|
| Lab |
Jan-Michael Peters' Lab
|
| Street address |
Campus-Vienna-Biocenter 1
|
| City |
Vienna |
| ZIP/Postal code |
1030 |
| Country |
Austria |
| |
|
| Platform ID |
GPL16159 |
| Series (2) |
| GSE41460 |
Wapl controls higher-order chromatin structure in interphase chromosomes (gene expression) |
| GSE41603 |
Wapl controls higher-order chromatin structure in interphase chromosomes |
|
