IP-seq of nuclease-protected RNA associated with Rbfox1(WT)/LASR and Rbfox1(F125A)/LASR

Rbfox1(WT) has a high affinity for GCAUG whereas Rbfox1(F125A) has a lower affinity for this motif. LASR associated with each protein was purified, nuclease-protected RNA was extracted, and libraries were prepared from both samples. A third control sample is the same protocol applied to cells that don't express FLAG-tagged Rbfox. Overall design: Purification protocol outlined in Damianov et al., 2016 was applied to Flp-In 293 Rbfox2 KO#7 line without any transgenes, with FLAG-Rbfox1(WT) line, and with FLAG-Rbfox1(F125A) . Next, RNA was extracted from protein complexes and libraries were prepared with a modified version of the iCLIP protocol outlined in Damianov et al 2016. RNA isolation procedure: Purified Rbfox/LASR complexes were digested with Proteinase K and RNA was extracted using phenol-chloroform and ethanol precipitation. The sample was then treated with TurboDNAse to remove any background DNA. cell or tissues: HEK293 Flp-In Rbfox2 KO#7