Protein-Centric Mapping of RNA-DNA Interactions with RedChIP

Noncoding RNAs (ncRNAs) are involved in a variety of processes in the cell nucleus, including transcriptional control, shaping 3D genome, and assembly/maintenance of functional nuclear compartments. However, the specific functions of most of the currently identified ncRNAs remain unclear. To gain further insight into the role of ncRNA in the eukaryotic genome functioning, it is important to determine genome-wide association patterns of various ncRNAs with chromatin. To address this question, a panel of RNA-DNA proximity ligation-based approaches have been developed, which have allowed deciphering RNA-chromatin interactions at the genome-wide level. An important drawback of all these techniques, however, is that they do not reveal the proteins involved in RNA-DNA interactions. In this chapter, we describe RedChIP, a method combining RNA-DNA ligation with chromatin immunoprecipitation to identify RNA-chromatin interactions mediated by a particular protein. We present a detailed protocol for RedChIP, focusing on the technical nuances and subtleties of the experimental procedure, and discuss algorithms for processing and analyzing the sequencing data. RedChIP can be used to identify RNAs associated with genomic regions occupied by any protein of interest, enabling disclosure of ncRNAs involved in recruiting various protein complexes to chromatin.