Integrated Analysis of Cross-Link-Ligation Data for the Detection of RNA 2D/3D Structures and Interactions In Vivo

The latest advancements in cross-linking- and proximity ligation-based methods have transformed the study of RNA structures and interactions in living cells. Despite this progress, sequencing data must be meticulously processed to extract the rich information. This presents distinct challenges, as noted in recent methods, e.g., Psoralen Analysis of RNA Interactions and Structures (PARIS) and Spatial 2'-Hydroxyl Acylation Reversible Cross-linking (SHARC). In this chapter, we detail the protocol used to analyze RNA sequencing data derived from cross-link-ligation experiments. We outline the strategies used to preprocess raw data, map to a reference genome, classify reads, and assemble duplex groups. Further, we show an example of analyzing structural data for the ribosomal RNA.