Integrated analysis of crosslink-ligation data for the detection of RNA 2D/3D structures and interactions in vivo

The latest advancements and implementation of crosslinking– and proximity-ligation based methods have transformed the study of RNA structure and interactions in living cells. Despite this progress, sequencing data must be meticulously processed to elucidate information on RNA. This presents distinct challenges, as noted in the Psoralen Analysis of RNA Interactions and Structures (PARIS) and Spatial 2′-Hydroxyl Acylation Reversible Crosslinking (SHARC) studies. In this chapter, we detail the process used to analyze RNA sequencing data derived from crosslink-ligation based experiments. We detail the strategies used to pre-process raw data, map to a reference genome, classify reads, and assemble duplex groups. Further, we show an example of analyzing structural data from the ribosomal RNA.