|
| Status |
Public on Oct 07, 2014 |
| Title |
Genomic discovery of potent chromatin insulators for human gene therapy |
| Organism |
Homo sapiens |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
|
| Summary |
This data includes regulatory factor profiling using ChIP-seq.
|
| |
|
| Overall design |
Cells were grown according to the approved ENCODE cell culture protocols. Cells were crosslinked with 1% formaldehyde, and the reaction was quenched by the addition of glycine. Fixed cells were rinsed with PBS, lysed in nuclei lysis buffer, and the chromatin was sheared to 200-500 bp fragments using Fisher Dismembrator (model 500). Sheared chromatin fragments were immunoprecipitated with specific polyclonal antibodies at 4 degrees C with gentle rotation. Antibody-chromatin complexes were washed and eluted. The cross linking in immunoprecipitated DNA was reversed and treated with RNase-A. Following proteinase K treatment, the DNA fragments were purified by phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation. 20-50 ng of ChIP DNA was end-repaired, adenine ligated to Illumina adapters was added, and then a Solexa library was made for sequencing. ChIP-seq affinity is directly reflected in raw tag density (Raw Signal), which is shown in the track as density of tags mapping within a 150 bp sliding window (at a 20 bp step across the genome). ChIP-seq affinity zones (HotSpots) were identified using the HotSpot algorithm described in Sabo et al. (2004). 1.0% false discovery rate thresholds (FDR 0.01) were computed for each cell type by applying the HotSpot algorithm to an equivalent number of random uniquely mapping 36mers. ChIP-seq affinity (Peaks) were identified as signal peaks within FDR 1.0% hypersensitive zones using a peak-finding algorithm.
|
| |
|
| Contributor(s) |
Liu M, Maurano MT, Wang H, Stamatoyannopoulos JA, Stamatoyannopoulos G |
| Citation(s) |
25580597 |
| |
| Submission date |
Sep 05, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Matthew T Maurano |
| E-mail(s) |
maurano@nyu.edu
|
| Organization name |
NYU School of Medicine
|
| Department |
Institute for Systems Genetics
|
| Lab |
Maurano
|
| Street address |
435 East 30th Street
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10016 |
| Country |
USA |
| |
|
| Platforms (1) |
| GPL11154 |
Illumina HiSeq 2000 (Homo sapiens) |
|
| Samples (4)
|
|
| Relations |
| BioProject |
PRJNA218089 |
| SRA |
SRP029596 |
Less...
More...