RBM33 eCLIP

Enhanced UV crosslinking immunoprecipitation (eCLIP) was performed to identify direct RNA targets of RBM33 Overall design: 20 million HCT116 or HEK293T cells were seeded in a 15 cm dish and reverse transfected with 40 µg pLJM1-EV or pLJM1-FH-RBM33 plasmid using Fugene HD (Promega). Media was changed 24 hours later. 48 hours after transfection, UV crosslinking was performed on ice at 250 nm (400 mJ/cm2) in a Spectrolinker XL-1500 (Spectronics). Cells were scraped in PBS, pelleted, snap-frozen in liquid nitrogen and stored at -80ºC until needed. Monoclonal FLAG M2 antibody (F1804, Sigma) conjugated to Dynabeads Protein G (Invitrogen) was used to immunoprecipitate FH-RBM33. For each cell line, duplicate size-matched input and IP samples were prepared and sequenced.