Progress and challenges in profiling protein–RNA and protein-associated RNA–RNA interactions

Comparison of chimeric ligation strategies and library preparation in RRI methods. Most methods now incorporate a T4 RNA ligase step without adaptors to encourage direct proximity ligation of base-paired RNAs. To further improve chimeric ligation efficiency and selectively enrich chimeric reads, hiCLIP and MARIO utilized a linker or biotinylated linker to bridge RNA duplexes. The 3′ cDNA adaptor can be added either to linear or circularized cDNA before PCR amplification. Additionally, irCLASH conjugates infrared dye and biotinylated adaptor for RNA visualization.