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| Status |
Public on Apr 17, 2017 |
| Title |
Cis Regulatory Element Scan by Tiling-deletion and Sequencing |
| Organisms |
Homo sapiens; synthetic construct |
| Experiment type |
Other
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
Here we report a highly scalable strategy for unbiased discovery and functional characterization of cis regulatory elements in the genome. These results demonstrate the utility of our cis element discovery strategy and reveal the dual function of gene promoters: they not only initiate transcription of their own genes, but could also control transcription of nearby genes.
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| Overall design |
As proof of principle, we focused on the 2Mbp POU5F1 locus (hg19 chr6:30120000-32150000) in hESCs with the goal to identify cis-regulatory elements involved in the transcriptional control of POU5F1. We first designed 11,570 sgRNA pairs that would introduce ~2kb-deletions in a highly nested manner with ~100bp step sizes. As negative controls, we included 424 ineffective oligos lacking the PAM sequence necessary for effective dsDNA breaks. Additionally, we also included 6 sgRNA pairs that target the eGFP coding sequence as positive controls. The dual CRISPR expressing cassettes were cloned into lentiCRISPRv2 vector, and the resulting lentiviral libraries were verified to be of high quality and capable of generating genomic deletions.
ATAC-seq is a widely used method to mapping the open chromatin landscape of the cells. In this study, we perfomed ATAC-seq to compare the open chromain patters in wild-type and two mutant clones with PRRC2A and MSH5 core promoter region deletions. The core promoters of PRRC2A and MSH5 have been identified as distal enhancer of POU5F1 and we want to test if and how the deletion of distal enhancer affect the open chromaitn patterns.
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| Contributor(s) |
Diao Y, Fang R, Li B, Meng Z, Marina RJ, Jung I, Guan K, Ren B |
| Citation(s) |
28417999 |
| Submission date |
May 02, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Bing Ren |
| Organization name |
University of California, San Diego School of Medicine
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| Street address |
9500 Gilman Dr., CMM-East, Admin Area/Rm 2071
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093 |
| Country |
USA |
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| Platforms (2) |
| GPL20301 |
Illumina HiSeq 4000 (Homo sapiens) |
| GPL21616 |
Illumina HiSeq 4000 (synthetic construct) |
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| Samples (23)
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| Relations |
| BioProject |
PRJNA320305 |
| SRA |
SRP074253 |
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