Global mapping of RNA G-quadruplexes (G4-RNAs) using G4RP-seq

Guanine-rich RNAs can fold into four-stranded structures, termed G-quadruplexes (G4-RNAs), and participate in a wide range of biological processes. Here we describe in detail a G4-RNA-specific precipitation (G4RP) protocol, which enables the transcriptomic profiling of G4-RNAs. The G4RP protocol consists of a chemical cross-linking step, followed by affinity capture with a G4-specific probe, BioTASQ. G4RP can be coupled with sequencing to capture a comprehensive global snapshot of folded G4-RNAs. This method can also be used to profile induced changes (i.e., through G4 ligand treatments) within the G4-RNA transcriptome. The entire protocol can be completed in 1–2 weeks and can be scaled up or down depending on the specific experimental goals. In addition to the protocol details, we also provide here a guide for optimization in different laboratory setups. Guanine-rich RNAs folded into G-quadruplex structures (G4-RNAs) are captured from cross-linked RNA using a G4-specific chemical probe, BioTASQ, and mapped globally by sequencing.