|
| Status |
Public on Nov 28, 2011 |
| Title |
Limb 4C Hoxd13 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Hoxd13 interacting sequences from E12.5 mouse Forelimbs/autopods
|
| Organism |
Mus musculus |
| Characteristics |
age: E12.5
developmental stage: embryo
tissue: Forelimb/ autopod
viewpoint: Hoxd13
genetic background: B6/CBA (F1)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Presumptive digits and forebrains were dissected from E12.5 mouse embryos, dissociated by collagenase treatment and fixed in 2 % formaldehyde for 10 minutes at room temperature. After cell lysis, nuclei were stored at -80°C. Pools of 16 autopods or 2 forebrains were digested with DpnII (New England Biolabs) and ligated in diluted conditions to promote intramolecular ligation events. DNA was purified by phenol/chloroform extraction followed by ethanol precipitation. Sequences ligated to the fragment of interest (interacting regions) were amplified by inverse PCR as described (Simonis et al., 2006), using the following primer pairs for each viewpoint: Hoxd13-F: 5'-GCACAGGCTCTTCGTCGT-3'; Hoxd13-R: 5'-CAAATTCGAGACAGCTCATCC-3'; Hoxd4-F: 5'-GTAGTTCAGAGCGGGGTTTG-3'; Hoxd4-R: 5'-GGCAGCTGGGATTAACACTTA-3'; IslandI-F: 5'-GGGACACGGTCCAATTTAAG-3'; IslandI-R: 5'-TGGAAAGGTCATGCGTGTT-3'; IslandIV-F: 5'-GGAATGACCAAGGGATACAAGA-3'; IslandIV-R: 5'-GGGTAAAGACTGCACAAGAGC-3'; Prox-F: 5'-GACGGCCACTCACATAATAAGA-3'; Prox-R: 5'-GGCTTGACAGCTGTGTAGTAGC-3'.. 200 ng of 4C template were amplified per reaction, using AmpliTaq DNA polymerase (Applied Biosystems). For each condition, 16 reactions were pooled and purified using Qiagen PCR clean-up kit.
|
| Label |
biotin
|
| Label protocol |
1 ug of 4C and input DNA was fragmented and labeled using GeneChip WT Double-Stranded DNA Terminal Labeling Kit (Affymetrix).
|
| |
|
| Channel 2 |
| Source name |
Input DNA from E12.5 mouse limbs
|
| Organism |
Mus musculus |
| Characteristics |
age: E12.5
developmental stage: embryo
tissue: Forelimb/ autopod
genetic background: B6/CBA (F1)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Presumptive digits and forebrains were dissected from E12.5 mouse embryos, dissociated by collagenase treatment and fixed in 2 % formaldehyde for 10 minutes at room temperature. After cell lysis, nuclei were stored at -80°C. Pools of 16 autopods or 2 forebrains were digested with DpnII (New England Biolabs) and ligated in diluted conditions to promote intramolecular ligation events. DNA was purified by phenol/chloroform extraction followed by ethanol precipitation. Sequences ligated to the fragment of interest (interacting regions) were amplified by inverse PCR as described (Simonis et al., 2006), using the following primer pairs for each viewpoint: Hoxd13-F: 5'-GCACAGGCTCTTCGTCGT-3'; Hoxd13-R: 5'-CAAATTCGAGACAGCTCATCC-3'; Hoxd4-F: 5'-GTAGTTCAGAGCGGGGTTTG-3'; Hoxd4-R: 5'-GGCAGCTGGGATTAACACTTA-3'; IslandI-F: 5'-GGGACACGGTCCAATTTAAG-3'; IslandI-R: 5'-TGGAAAGGTCATGCGTGTT-3'; IslandIV-F: 5'-GGAATGACCAAGGGATACAAGA-3'; IslandIV-R: 5'-GGGTAAAGACTGCACAAGAGC-3'; Prox-F: 5'-GACGGCCACTCACATAATAAGA-3'; Prox-R: 5'-GGCTTGACAGCTGTGTAGTAGC-3'.. 200 ng of 4C template were amplified per reaction, using AmpliTaq DNA polymerase (Applied Biosystems). For each condition, 16 reactions were pooled and purified using Qiagen PCR clean-up kit.
|
| Label |
biotin
|
| Label protocol |
1 ug of 4C and input DNA was fragmented and labeled using GeneChip WT Double-Stranded DNA Terminal Labeling Kit (Affymetrix).
|
| |
|
| |
| Hybridization protocol |
1μg of DNA was hybridzed per array for 16 hours at 45° at 60rpm using the Affymetrix hybridization kit.
|
| Scan protocol |
Arrays were scanned on an Affymetrix Scanner 3000 7G
|
| Description |
Hoxd13 interacting sequences from E12.5 mouse Forelimbs/autopods, Biological Rep. 1+2
|
| Data processing |
Data were quantile normalized within 4C/input replicate groups and scaled to medial feature intensity of 100 using TAS software (Affymetrix). For each genomic position, a data set was generated consisting of all (PM-MM) pairs mapping within a sliding window of 250 bp.
signal.bar and pvalue.bar are generated by TAS software (Affymetrix). signal.bar contains log2(IP/Input) fold changes for each probe in a region 2*sliding window+1. pvalue.bar contains -10log10 (IP/Input) for each probe in a region 2*sliding window+1.
|
| |
|
| Submission date |
Aug 25, 2011 |
| Last update date |
Nov 28, 2011 |
| Contact name |
Thomas Montavon |
| Organization name |
EPFL
|
| Street address |
SV 2843, Station 19
|
| City |
Lausanne |
| ZIP/Postal code |
1015 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL14183 |
| Series (2) |
| GSE31658 |
Spatial organization of chromatin at the HoxD locus in developing limbs and brain |
| GSE31659 |
HoxD locus in the developing limb, digit, and brain |
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