Flipper: An advanced framework for identifying differential RNA binding behavior with eCLIP data.

Motivation: Crosslinking and immunoprecipitation (CLIP) methods remain the gold standard for characterizing RNA binding protein (RBP) behavior. As a result, many researchers rely on CLIP to assess how treatments targeting RBPs alter binding patterns and regulatory activity. However, current tools for differential RBP binding analysis lack core features required for rigorous statistical inference, including proper normalization and appropriate handling of replicate experiments. Furthermore, existing approaches cannot adequately separate expression driven effects from true changes in RBP binding, complicating interpretation of differential analyses. Addressing these limitations is essential for producing reproducible and informative analyses of differential RBP binding. Results: Here we present Flipper, an application purpose built for the analysis of differential RBP binding. Flipper introduces several innovations that adapt the DESeq2 framework for robust differential analysis of eCLIP count data. These include integration of input controls to account for expression driven binding shifts, hierarchical normalization strategies that adjust for technical variation without confounding signal to noise ratios, and improved post-differential analysis tools. We demonstrate that Flipper exhibits high specificity when applied to real differential eCLIP data while also providing deeper biological insights. In addition, analyses of both real and simulated data indicate that Flipper achieves superior sensitivity and precision compared with existing approaches. Together, these results highlight Flipper as a robust and generalizable framework for differential RBP binding analysis.