Abstract
RNA-binding proteins are instrumental for post-transcriptional gene regulation, controlling all aspects throughout the lifecycle of RNA molecules. However, transcriptome-wide methods to profile RNA-protein interactions in vivo remain technically challenging and require large amounts of starting material. Herein, we present an improved library preparation strategy for crosslinking and immunoprecipitation (CLIP) that is based on tailing and ligation of cDNA molecules (TLC). TLC involves the generation of solid-phase cDNA, followed by ribotailing to significantly enhance the efficiency of subsequent adapter ligation. These modifications result in a streamlined, fully bead-based library preparation strategy, which eliminates time-consuming purification procedures and drastically reduces sample loss. As a result, TLC-CLIP displays unparalleled sensitivity, enabling the profiling of RNA-protein interactions from as few as 1000 cells. To demonstrate the effectiveness of TLC-CLIP, we profiled four endogenous RNA-binding proteins, showcasing its reproducibility and improved precision resulting from a higher occurrence of crosslinking-induced deletions. These deletions serve as an intrinsic quality metric and increase both specificity and nucleotide-resolution.