eCLIP Overlap Analysis and Validation

Overlap between eCLIP datasets A and B was determined by calculating the fraction of significant and reproducible peaks in dataset A that overlapped (by at least one base) a peak in dataset B, and vice versa the fraction of peaks in B that overlapped a peak in A, and taking the maximum of those fractions as the overall pairwise fraction overlap. Only datasets with at least 100 reproducible and significant peaks were used for this analysis. Gene Set Enrichment Analysis was performed using the GSEA software package [77 (link)]. RBP interaction data was obtained from the BioPlex 2.0 dataset [52 (link)].
IP-western validation was performed using HNNRPL (ab6106, Abcam), RBFOX2 (A300-864A, Bethyl), FMR1 (RN016P, Bethyl), AGGF1 (A303-634A, Bethyl), and TNRC6A (RN033P, MBLI) antibodies in UV crosslinked K562 cells. Immunoprecipitation in high-salt wash conditions was performed using standard eCLIP wash buffers, beads, and other reagents [18 (link)]. Low-salt co-immunoprecipitation conditions used identical conditions, except for lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% Sodium deoxycholate, and Protease Inhibitor cocktail (Promega)) and wash buffer (5 washes total in TBS + 0.05% NP-40). Westerns were probed with HNNRPL (ab6106, Abcam) primary antibody and TrueBlot secondary (Rockland).