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| Status |
Public on Apr 30, 2023 |
| Title |
Comprehensive RNA-Chromatin Interactome Reveals Global and Specific Regulatory Functions by Long Non-Coding RNAs |
| Organism |
Homo sapiens |
| Experiment type |
Other
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| Summary |
Long non-coding RNAs (lncRNAs) are an emerging class of regulatory molecules with a potentially broad-range of epigenomic regulatory functions. These functions are likely mediated by lncRNA-chromatin interactions either directly or indirectly through protein intermediates. Thus, comprehensive genomic mapping of all lncRNA targets describing the ‘RNA-chromatin interactome’ are critical for inferring lncRNA functions. Current methodologies for investigating lncRNA-chromatin interaction are one-RNA-at-a-time, and most inferred lncRNA functions were based on indirect “guilt-by-association”. To address these limitations, we devised an unbiased genome-wide strategy to identify all RNA interactions with chromatin by paired-end-tag sequencing (RICh-PET). We comprehensively characterized a human RNA-chromatin interactome and uncovered complex RNA-chromatin networks with many showing long-distance interactions predominantly at gene promoters. Further analysis revealed both global and specific transcriptional regulation by lncRNAs to genes involved in cancer biology, demonstrating the capacity of RICh-PET to concomitantly identify thousands of lncRNAs and chromatin targets in the human genome, thus significantly advancing the understanding of lncRNA functions.
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| Overall design |
Approximately 10^8 HeLa S3 cells were subjected to dual-crosslinking with 1% formaldehyde and 1.5 mM EGS. Cells were harvested and lysed. Complexes of lncRNA-chromatin with protein were sheared to fragments of approximately 500 bp, which then were biotinylated with EZlink Iodoacetyl-PEG2-Biotin (IPB). The streptavidin beads-bound lncRNA-chromatin-protein complexes were subjected to RICh-PET library construction. The DNA fragments present in streptavidin beads-bound chromatin were end-repaired using T4 DNA polymerase. The chromatin complexes were split into two aliquots, followed by the addition of RNA linkers with different barcode sets modified with biotin and containing flanking MmeI restriction sites, RNA linker “R1” (tube1) and RNA linker “R2” (tube2). Linkers to the RNA end were annealed, first-strand cDNA was synthesized, and relative DNA linkers “D1” (tube 1) and DNA linker “D2” (tube 2) were ligated. The two tubes were mixed together for proximity ligation. Chromatin-associated RNA fragments with linkers were subjected to second-strand cDNA synthesis. The cDNA-DNA PETs were extracted from the ligation products by MmeI digestion. Released biotinylated PETs were purified by streptavidin-coated magnetic beads, ligated to adaptors, and scaled up by PCR. Gel-purified amplicons of PET templates were sequenced using an Illumina® HiSeq 2500 with paired-end sequencing.
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| Contributor(s) |
Luo OJ, Zheng M, Li G |
| Citation missing |
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| Submission date |
Dec 06, 2013 |
| Last update date |
Apr 30, 2023 |
| Contact name |
Oscar J. Luo |
| E-mail(s) |
Oscar.Luo@jax.org
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| Organization name |
The Jackson Laboratory for Genomic Medicine
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| Street address |
10 Discovery Dr
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| City |
Farmington |
| State/province |
CT |
| ZIP/Postal code |
06030 |
| Country |
USA |
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| Platforms (1) |
| GPL16791 |
Illumina HiSeq 2500 (Homo sapiens) |
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| Samples (3) |
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| Relations |
| BioProject |
PRJNA230775 |
| SRA |
SRP033555 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE53052_CHH2430_JCHH2430_JCHH2431.DNA_RNA.txt.gz |
85.3 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data are available on Series record |
| Raw data are available in SRA |
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