 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Feb 18, 2019 |
| Title |
Determination of local chromatin interactions using a combined CRISPR and peroxidase APEX2 system |
| Organism |
Homo sapiens |
| Experiment type |
Other
|
| Summary |
The architecture and function of chromatin are largely regulated by local interacting molecules, such as transcription factors and noncoding RNAs. However, our understanding of these regulatory molecules at a given locus is limited because of technical difficulties. Here, we describe the use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and an engineered ascorbate peroxidase 2 (APEX2) system to investigate local chromatin interactions (CAPLOCUS). We showed that with specific small-guide RNA targets, CAPLOCUS could efficiently identify both repetitive genomic regions and single-copy genomic locus with high resolution. Genome-wide sequencing revealed known and potential long-range chromatin interactions for a specific single-copy locus. CAPLOCUS also identified telomere-associated RNA. CAPLOCUS, followed by mass spectrometry, identified both known and novel telomere-associated proteins in their native states. Thus, CAPLOCUS may be a useful approach for studying local interacting molecules at any given chromosomal location.
|
| |
|
| Overall design |
CAPLOCUS combined with NGS sequencing was carried out on a repetitive region on chromosome 13 (chr13: 112,930,173-112,968,847), a single-copy locus on chromosome 11 (chr11: 5,497,611-5,497,843), and sgGAL4 control in replicate.
In this method, with sgRNAs targeting a specific genomic region, local proteins were labeled with biotin and can be captured by M-280 Steptavidin Dynabeads. Thus, sgC13-IP and sgC11-IP were transfected with different sgRNAs targeting C13 region or C11 region, but both were pulled down with M-280 Steptavidin Dynabeads.
|
| |
|
| Contributor(s) |
Qiu W, Liu Y |
| Citation(s) |
30805613 |
| Submission date |
May 07, 2018 |
| Last update date |
May 21, 2019 |
| Contact name |
Yun Liu |
| Organization name |
Fudan University
|
| Street address |
Dongan Road 130
|
| City |
Shanghai |
| ZIP/Postal code |
200032 |
| Country |
China |
| |
|
| Platforms (1) |
| GPL9052 |
Illumina Genome Analyzer (Homo sapiens) |
|
| Samples (12)
|
|
| Relations |
| BioProject |
PRJNA464393 |
| SRA |
SRP144781 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE114133_RAW.tar |
1.2 Gb |
(http)(custom) |
TAR (of BW, NARROWPEAK) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
Less...
More...