Mapping Transcriptome-Wide and Genome-Wide RNA-DNA Contacts with Chromatin-Associated RNA Sequencing (ChAR-seq).
Methods in molecular biology (Clifton, N.J.)
2020; 2161: 115–42
RNAs play key roles in the cell as molecular intermediates for protein synthesis and as regulators of nuclear processes such as splicing, posttranscriptional regulation, or chromatin remodeling. Various classes of non-coding RNAs, including long non-coding RNAs (lncRNAs), can bind chromatin either directly or via interaction with chromatin binding proteins. It has been proposed that lncRNAs regulate cell-state-specific genes by coordinating the locus-dependent activity of chromatin-modifying complexes. Yet, the vast majority of lncRNAs have unknown functions, and we know little about the specific loci they regulate. A key step toward understanding chromatin regulation by RNAs is to map the genomic loci with which every nuclear RNA interacts and, reciprocally, to identify all RNAs that target a given locus. Our ability to generate such data has been limited, until recently, by the lack of methods to probe the genomic localization of more than a few RNAs at a time. Here, we describe a protocol for ChAR-seq, an RNA-DNA proximity ligation method that maps the binding loci for thousands of RNAs at once and without the need for specific RNA or DNA probe sequences. The ChAR-seq approach generates chimeric RNA-DNA molecules in situ and then converts those chimeras to DNA for next-generation sequencing. Using ChAR-seq we detect many types of chromatin-associated RNA, both coding and non-coding. Understanding the RNA-DNA interactome and its changes during differentiation or disease with ChAR-seq will likely provide key insights into chromatin and RNA biology.
View details for DOI 10.1007/978-1-0716-0680-3_10
View details for PubMedID 32681510