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| Status |
Public on Dec 31, 2020 |
| Title |
Transcriptome changes of Hela cells with blocking classical and alternative non-homologous end-joining (NHEJ) pathways |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Purpose: The recombinant endonucleases introduce double-strand breaks (DSBs) in genomic DNA at targeted sites and non-homologous end-joining (NHEJ) pathways repair the breaks introducing indels into the genome. To investigate whether there is another NHEJ pathway or if A-NHEJ and C-NHEJ are redundant, we examined NHEJ edits in HeLa cells.
Methods: To test among these possibilities, we engineered a NHEJ reporter system using targeted TALEN induced double strand DNA breaks. C-NHEJ was blocked in XRCC4(-/-) cells and A-NHEJ was blocked pharmacologically with mirin. To assess how NHEJ was maintained, differentially expressed genes were determined by RNA-Seq.
Results: When both C-NHEJ and A-NHEJ are blocked, edits were still observed. No changes in expression of DNA repair genes was observed. However, when both NHEJ pathways were blocked, genes from several pathways including DNA damage response, hypoxic response, chromatin packaging, p53 response and two genes that stimulate homologous recombination were differentially expressed.
Conclusions: NHEJ is maintained when both NHEJ pathways are blocked through a transcriptional response that must compensate for the inhibited steps in both pathways.
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| Overall design |
Overall design: Total eight samples separated in two experiments (biological duplicates) were sequenced, for profiling transcriptomes of Hela cells under four conditions – 1) Hela cells, 2) Hela cells with only blocking C-NHEJ pathway by XRCC4 knockout, 3) Hela cells with only blocking A-NHEJ pathway by mirin treatment, and 4) Hela cells with blocking both C-NHEJ and A-NHEJ pathways.
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| Contributor(s) |
Benjamin R, Schiller MR |
| Citation(s) |
35054780 |
| Submission date |
Aug 01, 2019 |
| Last update date |
Jan 31, 2022 |
| Contact name |
Martin Schiller |
| E-mail(s) |
martin.schiller@unlv.edu
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| Organization name |
University of Nevada, Las Vegas
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| Department |
Nevada Institute of Personalized Medicine
|
| Street address |
4505 S Maryland Pkwy
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| City |
Las Vegas |
| State/province |
NV |
| ZIP/Postal code |
89154 |
| Country |
USA |
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| Platforms (1) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
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| Samples (8)
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| Relations |
| BioProject |
PRJNA558142 |
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