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URL: https://dx.doi.org/DOI:10.17504/protocols.io.zv9f696

Authors: Ibrahim Ilik, Tugce Aktas, Daniel Maticzka, Rolf Backofen, Asifa Akhtar

Summary: 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 site 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.

Associated Publications: Ilik IA, Aktas T, Maticzka D, Backofen R, Akhtar A, FLASH: ultra-fast protocol to identify RNA–protein interactions in cells. Nucleic Acids Research 48(3). doi: 10.1093/nar/gkz1141

Affiliations: Max Planck Institute for Molecular Genetics, Berlin; Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Max Planck Institute for Molecular Genetics, Berlin; Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Bioinformatics Group, Department of Computer Science, University of Freiburg, Bioinformatics Group, Department of Computer Science, University of Freiburg ; Centre for Biological Signalling Studies (BIOSS), University of Freiburg, Max Planck Institute of Immunobiology and Epigenetics, Freiburg

External URL: https://doi.org/10.1093/nar/gkz1141

Version: 1

Publication Date: 2019

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