Mapping ligand-RNA interactions of thiamine analogues using transcriptome-wide sequencing

This chapter describes the use of transcriptome-wide sequencing to identify ligand-RNA interactions. Photoaffinity probes, based on small molecules of interest, can directly report on binding of ligands to their target RNA. Combined with pulldown enrichment experiments and next-generation sequencing (NGS), this chemical tool allows for transcriptome-wide screening to identify novel RNA targets. Here, we describe protocols to identify and validate ligand-RNA interactions using pulldown enrichments followed by transcriptome-wide sequencing or Reverse Transcription-quantitative PCR (RT-qPCR), and to investigate these interactions using Reverse-Transcription (RT) stop, Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) and in vitro transcription and translation (IVTT) assays.