|
| Status |
Public on Oct 23, 2013 |
| Title |
Brain 4C d13 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Brain
|
| Organism |
Mus musculus |
| Characteristics |
strain: Bl6/C57
tissue: Brain
age: E13.5
sample type: chromatin
|
| Treatment protocol |
Chromosome conformation capture on chip (4C) technique was performed as described (Hagège et al., 2007; Simonis et al., 2006). Caeca were dissected from E13.5 embryos, dissociated with collagenase (Sigma Aldrich/Fluka) and filtered through a 35 micron mesh to isolate single cells. Cells were fixed in formaldehyde 2% for 10 min at room temperature and reaction was quenched on ice with glycine. Cells were further lysated with 10 mM Tris pH 7.5, 10 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, PIC 1x to isolate nuclei. Nuclei from pools of fifty caecum or two brains were digested with DpnII (New England Biolabs) and ligated with T4 DNA ligase HC (Promega) in diluted conditions to promote intramolecular ligation. Sequences ligated to fragments of interest were amplified using previously described inversed PCR primers (Montavon et al., 2011) and AmpliTaq DNA polymerase (Applied Biosystems). 4C PCR was fragmented with apurinic / apyrimidinic endonucleases and uracil DNA glycosylases and labeled with terminal deoxynucleotidyl transferase using GeneChip WT Double- Stranded DNA Terminal Labeling Kit (Affymetrix). The profile of all different steps was analysed with a Bioanalyser. DNAs were hybridized to custom-made tiling arrays (Soshnikova and Duboule, 2009). Tiling arrays were processed according to manufacturer instructions.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Trizol
|
| Label |
biotin
|
| Label protocol |
standard Affymetrix protocol
|
| |
|
| Channel 2 |
| Source name |
gDNA
|
| Organism |
Mus musculus |
| Characteristics |
strain: Bl6/C57
age: E13.5
sample type: input
|
| Treatment protocol |
Chromosome conformation capture on chip (4C) technique was performed as described (Hagège et al., 2007; Simonis et al., 2006). Caeca were dissected from E13.5 embryos, dissociated with collagenase (Sigma Aldrich/Fluka) and filtered through a 35 micron mesh to isolate single cells. Cells were fixed in formaldehyde 2% for 10 min at room temperature and reaction was quenched on ice with glycine. Cells were further lysated with 10 mM Tris pH 7.5, 10 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, PIC 1x to isolate nuclei. Nuclei from pools of fifty caecum or two brains were digested with DpnII (New England Biolabs) and ligated with T4 DNA ligase HC (Promega) in diluted conditions to promote intramolecular ligation. Sequences ligated to fragments of interest were amplified using previously described inversed PCR primers (Montavon et al., 2011) and AmpliTaq DNA polymerase (Applied Biosystems). 4C PCR was fragmented with apurinic / apyrimidinic endonucleases and uracil DNA glycosylases and labeled with terminal deoxynucleotidyl transferase using GeneChip WT Double- Stranded DNA Terminal Labeling Kit (Affymetrix). The profile of all different steps was analysed with a Bioanalyser. DNAs were hybridized to custom-made tiling arrays (Soshnikova and Duboule, 2009). Tiling arrays were processed according to manufacturer instructions.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Trizol
|
| Label |
biotin
|
| Label protocol |
standard Affymetrix protocol
|
| |
|
| |
| Hybridization protocol |
standard Affymetrix protocol
|
| Scan protocol |
standard Affymetrix protocol
|
| Data processing |
Array data were normalized with input replicates and scaled to feature intensity of 100 using TAS software (Affymetrix). PM-MM pairs mapping within a sliding window of 250 bp was used.
bar files of normalized signals
|
| |
|
| Submission date |
Feb 15, 2013 |
| Last update date |
Oct 23, 2013 |
| Contact name |
Marion LELEU |
| Organization name |
EPFL
|
| Department |
School of Life Sciences
|
| Lab |
Laboratory of developmental genomics
|
| Street address |
EPFL-SV-ISREC-UPDUB- Station 19
|
| City |
Lausanne |
| ZIP/Postal code |
1015 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL14183 |
| Series (2) |
| GSE44357 |
Spatial organization of chromatin at the HoxD locus in developing caeca (4C tiling chip) |
| GSE45242 |
HoxD locus |
|
