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FLASH: ultra-fast protocol to identify RNA-protein interactions in cells

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Ilik,  Ibrahim Avsar
Quantitative RNA Biology (Tugce Aktas), Independent Junior Research Groups (OWL), Max Planck Institute for Molecular Genetics, Max Planck Society;

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Aktas,  Tugce
Quantitative RNA Biology (Tugce Aktas), Independent Junior Research Groups (OWL), Max Planck Institute for Molecular Genetics, Max Planck Society;

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Akhtar,  Asifa
Department of Chromatin Regulation, Max Planck Institute of Immunobiology and Epigenetics, Max Planck Society;

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Citation

Ilik, I. A., Aktas, T., Maticzka, D., Backofen, R., & Akhtar, A. (2020). FLASH: ultra-fast protocol to identify RNA-protein interactions in cells. Nucelic Acids Research, 48, e15. doi:10.1093/nar/gkz1141.


Cite as: http://hdl.handle.net/21.11116/0000-0005-7039-D
Abstract
Determination of the in vivo binding sites of RNA-binding proteins (RBPs) is paramount to understanding their function and how they affect different aspects of gene regulation. With hundreds of RNA-binding proteins identified in human cells, a flexible, high-resolution, high-throughput, highly multiplexible and radioactivity-free method to determine their binding sites has not been described to date. Here we report FLASH (Fast Ligation of RNA after some sort of Affinity Purification for High-throughput Sequencing), which uses a special adapter design and an optimized protocol to determine protein-RNA interactions in living cells. The entire FLASH protocol, starting from cells on plates to a sequencing library, takes 1.5 days. We demonstrate the flexibility, speed and versatility of FLASH by using it to determine RNA targets of both tagged and endogenously expressed proteins under diverse conditions in vivo.