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

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. Overall design: FLASH (Fast cloning of RNA After some Sort of affinity purification for High-throughput sequencing). This experiment investigates binding of two isoforms of the RNA-binding protein QKI (QKI-A and QKI-B) and a non-RNA-binding control (GFP), expressed using transiently transfected plasmids and performed with two biological replicates per condition.