Alternative splicing is a key mechanism for generating proteome diversity, however the mechanisms regulating alternative splicing are poorly understood. Using a genome-wide RNA interference screening strategy, we identified cleavage and polyadenylation specificity factor (CPSF) and symplekin (SYMPK) as cofactors of the well-known splicing regulator RBFOX2. To determine the role of CPSF in alternative splicing on a genome-wide level, we performed paired-end RNA sequencing (RNA-seq) to compare splicing events in control cells and RBFOX2 or CPSF2 knockdown cells. We also performed individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) to identify direct binding targets of RBFOX2 and CPSF2. Here, we describe the experimental design, and the quality control and data analyses that were performed on the dataset. The raw sequencing data have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE60392. © 2015.