PROJECT SUMMARY After transcription, RNA molecules undergo a number of processing steps that regulate the spliced sequence, RNA expression level, subcellular localization, and initiation and rate of translation, and are each controlled by the activity of RNA binding proteins (RBPs). RNA processing plays critical roles in normal development, and altered RNA processing has been shown to cause familial Spinal Muscular Atrophy, Amyotrophic Lateral Sclerosis, and multiple cancer types. Identifying the RNA targets of the RBP mutated in these diseases is a critical first step towards a mechanistic understanding of the disease and, ultimately, development of targeted therapeutics. However, standardized methods for identification of RBP targets transcriptome-wide were lacking, as they required large sample input amounts, had high experimental failure rates, and required high amplification leading to significant wasted sequencing and non-quantitative results. Recently we developed the enhanced CLIP-seq (eCLIP) methodology, which showed thousand-fold improved efficiency of generating high-throughput sequencing libraries from RBP profiling experiments and enabled highly robust and reproducible RBP target profiling through the incorporation of paired size-matched inputs. Here we will develop the eCLIP method for wide-spread use by the academic and commercial biotechnology industry in the following aims: 1. Develop an eCLIP kit, and perform optimization and stability testing to ensure robust data generation from a standardized kit format. 2. Perform alpha testing of a standardized eCLIP kit and protocol to validate experimental success and identify areas for further optimization. 3. Develop and validate standard conditions for various cell-types, tissues, and sample types (including flash-frozen versus fixed tissues, small sample amounts, and non-human samples). 4. Develop software tools for standard data processing and primary data analysis of eCLIP data, including integration with publicly available eCLIP data resources. Eclipse Bio is an ideal candidate to perform the aims described above due to our expertise in genomics and computational biology (particularly in the area of RNA processing), as well as in the starting of early-phase biology companies, make us ideal candidates to perform the research proposed above. The four aims above will take eCLIP from an academic research method to one that can be standardly performed by biomedical researchers in academia as well as commercial entities, without requiring extensive expertise in RNA biology. The development of standardized commercial kits to profile DNA binding protein targets by ChIP-seq rapidly led to large-scale adoption of this technique, and has provided significant benefits to studies of DNA binding proteins involved in various diseases; development of an eCLIP kit will similarly enable studies of RNA binding proteins. Additionally, our location in San Diego proximal to numerous academic research institutes (including UCSD and the Salk Institute) as well as biotechnology companies will provide scientific and commercialization expertise and assistance. [[currently 4 lines over 30 line limit]]