Overview

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Protein-RNA Interaction Profiling Sequencing (PIP-Seq) is a specific approach for profiling gene expression at the single-cell level using ribonuclease-based footprinting followed by high-throughput sequencing to evaluate both protein-bound RNA sequences and RNA secondary structure. It is used to investigate the interaction between proteins and RNA on a genome-wide scale at the single-cell level to gain the insights of cellular mechanisms, and gene regulation. PIP-seq utilizes single- and double-stranded RNA-specific nucleases in the absence or presence of proteins to infer RNA secondary structure and RNA protein interaction sites. This technique includes the following steps:
 

1. Crosslink cells or tissue to capture the protein-RNA interaction at their native states.

2. Immunoprecipitation to capture the proteins of interest. 

3. Isolation of RNA bound to the proteins of interest. 

4. cDNA construction, Library preparation and sequencing, followed with data analysis.

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Applications

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• Identify the binding sites of RNA-binding proteins (RBPs) and understand their role in post-transcriptional regulation.

• Uncover how gene expression is regulated at the transcriptional and post-transcriptional levels.

• Map the Genome-wide Interactions between RNA and RNA binding proteins.

• Reveal the potential therapeutic targets by identify the protein-RNA interaction in pharmaceutical research.