|
| Status |
Public on Sep 15, 2008 |
| Title |
Gli3Genome_mm8_3 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
FLAG ChIP DNA from E11.5 Prrx1Cre;Gli3T-Flag limb buds
|
| Organism |
Mus musculus |
| Characteristics |
Age: E11.5 mouse limb buds
Amplification method: LMPCR
Antibody: FLAG M2 mouse monoclonal
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Rosa Gli3TFLAG c/c females mice were crossed with Prrx1Cre homozygous male drivers and litters were taken at E11.5. Embryos (generally 9) were dissected and forelimbs and hindlimbs were dissected into PBS, trypsinized in 300μl trypsin-EDTA for 5 minutes at 37º, inactivated by adding 700μl of DMEM with 10% calf serum, and cross-linked with 1% formaldehyde for 30 minutes. ChIP and LMPCR was performed according to our previous protocol (Vokes et al., 2007) with the following modifications. Chromatin was sonicated in 2ml of buffer 3 using 10x30 second pulses on a Branson 450 Sonifier (3 pulses on setting 4, then 7 on setting 5). For whole genome arrays, three ChIP reactions were set up in parallel with 500μl each (with 15μl of each biological sample reserved for an input control) – single promoter arrays used a single ChIP sample. At the final step, the samples were pooled and enrichment was validated using qPCR. For LMPCR, the pooled samples were divided into three and processed in parallel using 0.25mM dATP, dCTP, dGTP, 0.2mM dTTP and 0.05mM dUTP with 27 total cycles and then re-pooled after PCR cleanup. Each pooled set of reaction yielded >38μg of amplified sample. The linearity of amplification was confirmed by comparing qPCR values with the values from the unamplified samples.
|
| Label |
Biotin
|
| Label protocol |
Samples (37.5-44μg of DNA) were enzymatically fragmented and labeled using the WT Labeling Kit (Affymetrix) to add biotinylated compound to the ends of the fragments
|
| |
|
| Channel 2 |
| Source name |
Input DNA from E11.5 Prrx1Cre;Gli3T-Flag limb buds
|
| Organism |
Mus musculus |
| Characteristics |
Age: E11.5 mouse limb buds
Amplification method: LMPCR
Input DNA
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Rosa Gli3TFLAG c/c females mice were crossed with Prrx1Cre homozygous male drivers and litters were taken at E11.5. Embryos (generally 9) were dissected and forelimbs and hindlimbs were dissected into PBS, trypsinized in 300μl trypsin-EDTA for 5 minutes at 37º, inactivated by adding 700μl of DMEM with 10% calf serum, and cross-linked with 1% formaldehyde for 30 minutes. ChIP and LMPCR was performed according to our previous protocol (Vokes et al., 2007) with the following modifications. Chromatin was sonicated in 2ml of buffer 3 using 10x30 second pulses on a Branson 450 Sonifier (3 pulses on setting 4, then 7 on setting 5). For whole genome arrays, three ChIP reactions were set up in parallel with 500μl each (with 15μl of each biological sample reserved for an input control) – single promoter arrays used a single ChIP sample. At the final step, the samples were pooled and enrichment was validated using qPCR. For LMPCR, the pooled samples were divided into three and processed in parallel using 0.25mM dATP, dCTP, dGTP, 0.2mM dTTP and 0.05mM dUTP with 27 total cycles and then re-pooled after PCR cleanup. Each pooled set of reaction yielded >38μg of amplified sample. The linearity of amplification was confirmed by comparing qPCR values with the values from the unamplified samples.
|
| Label |
Biotin
|
| Label protocol |
Samples (37.5-44μg of DNA) were enzymatically fragmented and labeled using the WT Labeling Kit (Affymetrix) to add biotinylated compound to the ends of the fragments
|
| |
|
| |
| Hybridization protocol |
Approximately 8.25μg of DNA was hybridzed per array using the Affymetrix hybridization kit. For these 7 array sets, 4 arrays were hybridized for 16 hours at 45° at 60rpm using an Affymetrix hybridization oven. The probe was removed from these arrays and reused to hybridize the remaining three arrays.
|
| Scan protocol |
Arrays were scanned on an Affymetrix Scanner 3000 7G
|
| Description |
GLI3 ChIP Biological Rep 1-3, chip C
|
| Data processing |
Data was quantile normalized and analyzed with TileMap. Log2(IP/Input) fold changes (fc) and TileMap moving average (ma) statistics were reported in BAR files.
|
| |
|
| Submission date |
Apr 04, 2008 |
| Last update date |
Jan 29, 2022 |
| Contact name |
Steven A. Vokes |
| E-mail(s) |
svokes@austin.utexas.edu
|
| Organization name |
University of Texas at Austin
|
| Department |
Molecular Biosciences
|
| Street address |
2500 Speedway, STOP A5000
|
| City |
Austin |
| State/province |
Texas |
| ZIP/Postal code |
78757 |
| Country |
USA |
| |
|
| Platform ID |
GPL6446 |
| Series (2) |
| GSE11062 |
Genome-Wide Gli3 Binding Sites in the E11.5 Mouse Limb Bud |
| GSE11077 |
A Genome-Scale Analysis of the Cis-Regulatory Circuitry Underlying Hedgehog Mediated Patterning of the Mammalian Limb |
|
