|
| Status |
Public on Jun 06, 2009 |
| Title |
H3K27me3_LF2_10d-EB |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
H3K27me3 ChIP DNA from LF2 10d EB ES cells
|
| Organism |
Mus musculus |
| Characteristics |
gender: female
strain: 129/Ola
antibody: H3K27me3
|
| Treatment protocol |
grown to embryoid bodies for 10 days
|
| Growth protocol |
ES cells grown on gelatin coated plates with LIF and ES grade serum
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was fixed in 1% formaldehyde for 10 minutes, sheared to 200-700bp using a Bioruptor (Diagenode). It was immunoprecipitated using the indicated antisera. Genomic DNA was isolated from the precipitated material as well as from the sheared chromatin input by phenol extraction and ethanol precipitation. The ChIP material was amplified using two rounds of lineair T7 amplification. All further details were performed as indicated by http://www.nimblegen.com/products/lit/lit.html
|
| Label |
Cy5
|
| Label protocol |
1 µg ChIP DNA (or cDNA) was directly labeled by Klenow (New England Biolabs) random priming with Cy5 nonamers (ChIP DNA isolated from normal rabbit IgG-immunoprecipitated cells) per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html). 1 µg genomic DNA was labeled similarly using Cy3 and used as Input.
|
| |
|
| Channel 2 |
| Source name |
Input genomic DNA from LF2 10d EB ES cells
|
| Organism |
Mus musculus |
| Characteristics |
reference: input genomic DNA
|
| Treatment protocol |
grown to embryoid bodies for 10 days
|
| Growth protocol |
ES cells grown on gelatin coated plates with LIF and ES grade serum
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was fixed in 1% formaldehyde for 10 minutes, sheared to 200-700bp using a Bioruptor (Diagenode). It was immunoprecipitated using the indicated antisera. Genomic DNA was isolated from the precipitated material as well as from the sheared chromatin input by phenol extraction and ethanol precipitation. The ChIP material was amplified using two rounds of lineair T7 amplification. All further details were performed as indicated by http://www.nimblegen.com/products/lit/lit.html
|
| Label |
Cy3
|
| Label protocol |
1 µg ChIP DNA (or cDNA) was directly labeled by Klenow (New England Biolabs) random priming with Cy5 nonamers (ChIP DNA isolated from normal rabbit IgG-immunoprecipitated cells) per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html). 1 µg genomic DNA was labeled similarly using Cy3 and used as Input.
|
| |
|
| |
| Hybridization protocol |
The labeled ChIP DNA (Cy5) and genomic DNA (Cy3) 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 simultaniously with the Cy5 and Cy3 labeled material 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 |
ChIP-chip LF2 ES cells
|
| Data processing |
Arrays were processed using Nimblegen's standard protocol for Nimblescan 2.4 ChIP data extraction; Raw data was used for visualisations. Each feature on the array has a corresponding scaled log2-ratio. This is the ratio of the input signals for the experimental and test samples that were co-hybridized to the array. The log2-ratio is computed and scaled to center the ratio data around zero. Scaling is performed by subtracting the bi-weight mean for the log2-ratio values for all features on the array from each log2-ratio value.
|
| |
|
| Submission date |
Jun 04, 2009 |
| Last update date |
Jun 05, 2009 |
| Contact name |
H.G. Stunnenberg |
| E-mail(s) |
h.stunnenberg@ncmls.ru.nl
|
| Phone |
+31-24-3610520
|
| Organization name |
Radboud University
|
| Department |
Department of Molecular Biology (274)
|
| Street address |
Geert Grooteplein 28
|
| City |
Nijmegen |
| ZIP/Postal code |
6525 GA |
| Country |
USA |
| |
|
| Platform ID |
GPL8656 |
| Series (2) |
| GSE15883 |
High resolution analysis of epigenetic changes associated with X inactivation, ChIP-chip |
| GSE15884 |
High resolution analysis of epigenetic changes associated with X inactivation |
|
