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

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.

SUBMITTER: Ilik IA

PROVIDER: S-EPMC7026646 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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

Ilik Ibrahim Avsar IA Aktas Tugce T Maticzka Daniel D Backofen Rolf R Akhtar Asifa A

Nucleic acids research 20200201 3

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-thr ...[more]

PMID: 31802123

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

Project description: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. We demonstrate the flexibility, speed and versatility of FLASH by using it to determine binding sites of both tagged and endogenously expressed proteins under diverse conditions. The entire FLASH protocol, starting from cells-on-plates to a sequencing library, takes 1.5 days.

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

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