| Autor: |
Weidao Zhang, Min Tang, Lin Wang, Ping Zheng, Bo Zhao |
| Jazyk: |
angličtina |
| Rok vydání: |
2023 |
| Předmět: |
|
| Zdroj: |
Bio-Protocol, Vol 13, Iss 21 (2023) |
| Druh dokumentu: |
article |
| ISSN: |
2331-8325 |
| DOI: |
10.21769/BioProtoc.4869 |
| Popis: |
Fork stability is key to genome DNA duplication and genetic integrity. Long non-coding RNAs (LncRNAs) may play vital roles in fork stabilization and chromatin remodeling. Existing techniques such as NCC-RNA sequencing are useful to identify LncRNAs on nascent chromatin DNA. However, there is still a lack of methods for LncRNAs purification directly from replicative forks, hindering a deep understanding of the functions of LncRNAs in fork regulation. Here, we provide a step-by-step protocol named iROND (isolate RNAs on nascent DNA). iROND was developed and modified from iPOND, a well-known method for purifying fork-associated proteins. iROND relies on click chemistry reaction of 5'-ethynyl-2'-deoxyuridine (EdU)-labeled forks and biotin. After streptavidin pull down, fork-associated LncRNAs and proteins are purified simultaneously. iROND is compatible with downstream RNA sequencing, qPCR confirmation, and immunoblotting. Integrated with functional methods such as RNA fluorescent in situ hybridization (RNA FISH) and DNA fiber assay, it is feasible to screen fork-binding LncRNAs in defined cell lines and explore their functions. In summary, we provide a purification pipeline of fork-associated LncRNAs. iROND is also useful for studying other types of fork-associated non-coding RNAs.Key features• Purify long non-coding RNAs (LncRNAs) directly from replication forks.• Connects to RNA sequencing for screening easily.• Allows testing various genotoxic stress responses.• Provides LncRNA candidate list for downstream functional research.Graphical overviewSchematic overview of isolate RNAs on nascent DNA (iROND) protocol. Cells were pulse-labeled with 5'-ethynyl-2'-deoxyuridine (EdU) for 10 min before paraformaldehyde fixation. EdU-positive forks were ligated with biotin through Click-IT chemistry reaction. Genomic DNA was ultrasonically cracked and crosslinked with streptavidin for pulling down. Both RNA and protein components were purified. RNA components were used for downstream RNA sequencing and qPCR validation. Protein components were used for immunoblotting to evaluate binding dynamics of fork-associated proteins such as helicase, topoisomerase, and DNA polymerases. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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