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Protein Interaction Profile Sequencing (PIP‐seq)

Every eukaryotic RNA transcript undergoes extensive post‐transcriptional processing from the moment of transcription up through degradation. This regulation is performed by a distinct cohort of RNA‐binding proteins which recognize their target transcript by both its primary sequence and secondary structure. Here, we describe protein interaction profile sequencing (PIP‐seq), a technique that uses ribonuclease‐based footprinting followed by high‐throughput sequencing to globally assess both protein‐bound RNA sequences and RNA secondary structure. PIP‐seq utilizes single‐ and double‐stranded RNA‐specific nucleases in the absence of proteins to infer RNA secondary structure. These libraries are also compared to samples that undergo nuclease digestion in the presence of proteins in order to find enriched protein‐bound sequences. Combined, these four libraries provide a comprehensive, transcriptome‐wide view of RNA secondary structure and RNA protein interaction sites from a single experimental technique. © 2016 by John Wiley & Sons, Inc.