Transcriptome-wide mapping of RNA:protein interactions of SRSF7 variants in P19 cells by iCLIP and piCLIP

We used P19 cells that overexpress GFP-tagged SRSF7 by 3.4-fold and expression-matched SRSF7 mutants that either lack a functional Zinc-knuckle domain or a part of their RS-domain and compared the extent and pattern of binding to mRNAs. For this we performed iCLIP using anti-GFP antibodies. We also performed iCLIP from monosomal and polysomal fractions. In addition we performed iCLIP using anti-SRSF7 antibodies to compare the binding patterns of SRSF7-GFP, endogenous SRSF7 protein and its truncated SRSF7_RRM variant. Overall design: For each GFP-tagged SRSF7 protein, 2-3 biological replicates were subjected to the iCLIP procedure with UV-crosslinking at 254 nm. For IPs either an anti-GFP antibody was used or an anti-SRSF7 antibody.