Analysis of Nucleolin and PTBP1 RNA targets before after Sciatic nerve injury

We previously showed that importin beta1 local translation in injured axons is a key event in injury-signaling and regeneration (Perry et al., 2012). Next, we showed that importin beta1 is localize to axons through a localization motif in its 3'UTR and that this motif is bound by the RNA binding proteins Nucleolin and PTBP1. In this study, we asked which other RNA targets are bound by these two RNA binding proteins in naive Sciatic nerves and how are they changing 7 days after injury. Overall design: We immunoprecipitated Nucleolin and PTBP1 from naive or injured Sciatic nerves (7 days after injury), isolated the bound RNA and analyzed it by RNA sequencing. As controls, we pre-incubated the antibodies used for immunoprecipitation with their respective blocking peptides, so as to block their ability to bind the target proteins. We also sequenced as reference a fraction of the input material from each sample. We pooled 10 Sciatic nerves per sample and repeated this experiment in 3 independent biological repeats.