EMSA was performed using IRDye-700 labeled 10 nt poly(A) and GC (GGCCCCCCGG) RNA oligos (Integrated DNA Technologies). PRPF39 was added to the RNA sample (10 nM) in binding buffer (50 mM Tris pH 7, 150 mM NaCl, 20% glycerol, 3 mM MgCl2, 1 mM DTT) to final concentrations of 270, 617, 964, 1311, 1658, 2005, 2352, and 2700 nM. The concentration range for PRPF39 NTD and CTD were 10, 30, 90, 270, 810, 2430, and 7290 nM. All reactions had a final volume of 5 µL including murine RNA inhibitor (New England Biolabs) and were incubated at 30°C for 15 min. The EMSA samples were analyzed on a nondenaturing PAGE (5% acrylamide; 37.5:1 acrylamide: N,N-methylene-bis-acrylamide [BioRad]) with 0.2× TBE buffer (26 mM Tris pH 7.5, 9 mM boric acid, 0.5 mM EDTA) and 2.5% glycerol (Liu et al. 2016 (link)). The gels were prerun at 100 V for 40 min before loading sample and subsequently electrophoresed at 100 V for 65 min. The RNA was visualized using an Odyssey infrared imaging system (LiCor). The band intensities were measured using AzureSpot (Azure Biosystems) analysis software. After background subtraction, the fraction of RNA bound was calculated, fitted with the Hill equation, and graphed using Prism software (GraphPad).