RNA-binding proteins (RBPs) interact with RNA molecules to form ribonucleoprotein complexes (RNPs), which affect the processing and/or function of RNA. For ENCODE 3, of the estimated 1,542 human RBP-encoding genes, up to 356 were studied in two commonly used cell lines (K562 and HepG2) using up to five different assays, generating a total of 1,223 datasets1. Enhanced crosslinking and immunoprecipitation followed by sequencing (eCLIP) was performed for 120 RBPs in K562 cells and 103 RBPs in HepG2 cells (73 RBPs in both cell types) to map transcriptome-wide RNA binding sites of RBPs. Overlaying eCLIP peaks onto GENCODE transcript annotations enabled the researchers to classify RBPs into six RNA types, some of which overlapped with well-known functions. RNA-seq in cells with small-hairpin RNA (shRNA) or CRISPR-mediated knockdown of RBPs revealed specific functions of RBPs in RNA stability and splicing. Integration with eCLIP targets indicated that only a limited set of RNAs showed both altered expression and eCLIP enrichment for the same RBP, indicating that many of the widespread differences in gene expression observed upon RBP knockdown were the result of indirect effects. To identify consensus RNA-binding sequences and structural motifs within RBP-bound RNAs, RNA Bind-N-Seq (RBNS) was carried out in vitro. Highly enriched kmers of five nucleotides were identified for the 78 tested RBPs, which could be clustered into one or more motifs. In most cases, the top 5mer of an RBP was enriched in the eCLIP peaks for this RBP, showing good agreement between in vitro and in vivo binding behaviour. Chromatin immunoprecipitation with sequencing (ChIP–seq) of RBPs yielded reproducible peaks in 52% of RBPs assayed in HepG2 cells and 64% of RBPs assayed in K562 cells, showing that most of these peaks were enriched at euchromatin and suggesting widespread interactions between RBPs and actively transcribed regions of the genome. With the help of immunofluorescence, diverse subcellular distribution patterns of RBPs were revealed, with localization to specific compartments being associated with binding to distinct RNA targets (for example, RBPs that localized to nucleoli preferentially bound 45S precursor rRNAs and small nucleolar RNAs). The in vivo binding affinities of 150 RBPs reported here represent about 10% of proteins that have been predicted to interact with RNA and lay important groundwork for understanding the complexity of post-transcriptional regulation in humans.