Pre-mRNA splicing is a highly regulated process that generates the mature mRNA which can then be translated into a protein. Alternative splicing (AS) allows creation of multiple mature mRNA isoforms from the same pre-mRNA, which dramatically increases the proteomic diversity and finetunes numerous cellular processes. With the advent of Next-Generation Sequencing (NGS) and its ever-falling costs in the last two decades, high-throughput quantification of all transcripts of a biological sample became first viable and nowadays routine. The studies presented in this dissertation combine biochemical and bioinformatical methods to elucidate how AS is regulated by exogenous and endogenous factors. They demonstrate how the nearly 30 petabytes of publicly available NGS data can be leveraged to identify global patterns, to perform highly integrative analyses and to deepen our understanding of disease-causing mutations. We identified AS to be controlled by body temperature in mammals, first for a single target and then globally for numerous examples. Since body temperature of mammals varies depending on their circadian rhythm, these isoforms are generated in a circadian-like fashion. In addition, we found Cdc-like kinase and temperature-dependent SR protein phosphorylation to be the driving force behind this mechanism. We then discovered that the nonsense-mediated decay pathway is often triggered by temperature-dependent isoforms. This leads to cycling expression of the affected genes, a phenomenon that was previously described but poorly understood. Furthermore, we discovered dramatically altered early secretory pathway capacities depending on Sec16a AS. We followed this up with a thorough bioinformatical analysis to identify further isoforms that influence transport processes. Indeed, we found hundreds of AS events that modulate the whole secretory pathway. An effect of these targets on membrane trafficking was validated in vivo. We demonstrated that these adaptations of the secretory pathway are highly tissue-specific and often induced in dynamic differentiation and activation processes. Finally, we interrogated two disease associated mutations in the (RNA binding) splicing factor U2AF35. Although these mutations have been annotated to be missense mutations and only change one amino acid, we determined that one of them leads to the creation of a cryptic splice site and therefore a four amino acid deletion. We investigated these mutant proteins in detail and could describe unique RNA binding characteristics. Lastly, we confirmed usage of the cryptic splice site and an effect of the resulting protein in patients carrying this specific mutation. All these findings heavily relied on the use of RNA-sequencing data analysis. Such studies illustrate the power of using NGS datasets and integrating them into biochemical research. Using complementary and interdisciplinary approaches like the ones presented here accelerates research, saves time and money and allows to gain novel insights unimaginable a decade ago.