A majority of human genes undergo extensive alternative splicing (AS), producing multiple RNA and protein isoforms form a single gene locus. AS is tightly regulated in different tissues, and such regulation is critical for normal physiology of distinct tissues as AS plays key roles to shape the cellular proteomic profile. Like transcription regulation, splicing is generally regulated by RNA cis-elements that recruit protein trans-factors to either activate or repress the use of adjacent splice sites. In this proposal, we will develop a novel approach for the high throughput identification of splicing regulatory cis-elements in a wide variety of cell types. This approach will combine the unique biology of adeno-associate virus (AAV) and the recent advance in parallel sequencing technology to achieve a fast identification of all splicing regulatory cis-elements. A large set of tissue specific splicing regulatory cis-elements will be obtained with our new approach, and we will further determine how these cis-elements regulate splicing inside a cell with both computational and experimental methods. We will further integrate such information into a splicing simulation algorithm to model the regulation of tissue specific splicing. We will first apply this approach in primary hepatocytes since liver has extensive AS events that probably play important roles in regulating liver functions. Collectively, these studies will allow us to determine a new set of rules for how different sequence elements regulate tissue specific splicing.