RNA sequencing of Flp-In 293 Rbfox2 KO#7 parental, Rbfox1(WT), and Rbfox1(F125A) expressing cells

How does Rbfox's binding to its canonical motif through its RRM contribute to its splicing activity? Splicing changes will be assessed in cells that don't express Rbfox versus cells that express wildtype Rbfox1, and cells that express the RNA binding mutant Rbfox1(F125A). Overall design: FLAG-Rbfox1(WT) and FLAG-Rbfox1(F125A) were integrated into the Flp-In locus in Flp-In 293 Rbfox2 KO#7 parental line. All three lines were treated with doxycyline for 48 hrs to induce expression of the transgenes. Cells were then harvested and both RNA and protein were extracted. The protein levels were assessed with immunoblotting and were similar between FLAG-Rbfox1(WT) and FLAG-Rbfox1(F125A). RNA was extracted and submitted to UNGC sequencing core for polyA RNA-seq. Note that each line was grown in triplicate and samples for RNA-seq were submitted for each replicate resulting in a total of 9 samples sequenced: 3 replicates of Flp-In 293 Rbfox2 KO#7 line, 3 replicates of Flp-In 293 Rbfox2 KO#7 F-Rbfox1(WT) line, and 3 replicates of Flp-In 293 Rbfox2 KO#7 F-Rbfox1(F125A) line.