The authors have developed an enhanced CLIP (eCLIP) protocol to improve the efficiency and specificity of identifying RNA-binding protein (RBP) binding sites. eCLIP reduces the required amplification by ~1,000-fold, decreases PCR duplicate reads by ~60%, and maintains single-nucleotide binding resolution. By simplifying the generation of paired IgG and size-matched input controls, eCLIP improves specificity in the discovery of authentic binding sites. The authors performed 102 eCLIP experiments for 73 diverse RBPs in HepG2 and K562 cells, demonstrating that eCLIP enables large-scale and robust profiling with amplification and sample requirements similar to those of ChIP-seq. eCLIP enables integrative analysis of diverse RBPs, revealing factor-specific profiles, common artifacts in CLIP, and RNA-centric perspectives on RBP activity. The improved eCLIP protocol provides a robust framework for large-scale transcriptome-wide binding map generation for RBPs, enhancing the understanding of their roles in cellular physiology.The authors have developed an enhanced CLIP (eCLIP) protocol to improve the efficiency and specificity of identifying RNA-binding protein (RBP) binding sites. eCLIP reduces the required amplification by ~1,000-fold, decreases PCR duplicate reads by ~60%, and maintains single-nucleotide binding resolution. By simplifying the generation of paired IgG and size-matched input controls, eCLIP improves specificity in the discovery of authentic binding sites. The authors performed 102 eCLIP experiments for 73 diverse RBPs in HepG2 and K562 cells, demonstrating that eCLIP enables large-scale and robust profiling with amplification and sample requirements similar to those of ChIP-seq. eCLIP enables integrative analysis of diverse RBPs, revealing factor-specific profiles, common artifacts in CLIP, and RNA-centric perspectives on RBP activity. The improved eCLIP protocol provides a robust framework for large-scale transcriptome-wide binding map generation for RBPs, enhancing the understanding of their roles in cellular physiology.