Project Summary The objective of the ENCORE (Encyclopedia of RNA Elements) project is to develop a foundational, functional map of protein-RNA interactions of RNA binding proteins (RBPs) encoded in the human genome, and the RNA elements they bind to across the transcriptome. These RNA elements, when expressed, form the basis of co- and post-transcriptional regulation of human genes. Our strategy consists of developing and integrating a physical map of 400 new RBPs in two different human cell lines with transcriptome-wide measurements of the effects of depleting these RBPs. Over the past 9 years, our group has established highly efficient data production workflows of experimental methods that will enable us to immediately expand these datasets further, which form a crucial and missing link to decipher the mechanisms of post-trancriptional regulation and how these impact genetic variation and disease etiology. When combined with the data we generated over the past 9 years, these efforts will culminate in a comprehensive map of the functional RNA elements recognized by essentially all RBPs expressed in two human cell lines, representing approximately half of the known complement of human RBPs. ENCORE will continue developing and integrating a physical map of hundreds of RBPs in two different human cell lines with transcriptome-wide measurements of the effects of depleting these RBPs. In addition, we will provide training and outreach to establish ENCORE annotations as the standard reference for co- and post-transcriptional research and clinical genomics efforts in the long-term. In summary, the data we will produce in this project will enable a more systematic and comprehensive understanding of the role of RBPs and RNA biology in the contribution to human biology and disease.