Identification of mRNA subsets interacting to RNA binding proteins in regenerating axons

The goal of this study is to compare the mRNA interactome of different RBPs in regenerating axons utilizing RNA-immunoprecipitation (RIP). Interacting mRNAs to each target RBPs were co-immunoprecipitated from axoplasm of sciatic nerve, injured 7 days ago. Interactome of each RBPs were identified by Next-generation sequencing (NGS). Overall design: SD rats underwent crush injury of the sciatic nerve. 7 days after injury, proximal sciatic nerve was excised and axoplasm was extruded with detergent free, isotonic buffer. After clarification with centrifugation (20,000 xg, 20 min) and subsequent preclearing with Protein A/G dynabeads, RBP and associated mRNA was precipitated by specific antibodies against La/SSB, hnRNP H1, hnRNP F, hnRNP K and FLAG (n=3 respectively) and Protein A/G dynabeads.