Mapping Transcriptome-Wide and Genome-Wide RNA-DNA Contacts with Chromatin-Associated RNA Sequencing (ChAR-seq).

Mapping Transcriptome-Wide and Genome-Wide RNA-DNA Contacts with Chromatin-Associated RNA Sequencing (ChAR-seq). Methods Mol Biol. 2020;2161:115-142 Authors: Limouse C, Jukam D, Smith OK, Fryer KA, Straight AF Abstract 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...
Source: Mol Biol Cell - Category: Molecular Biology Authors: Tags: Methods Mol Biol Source Type: research