Neural Stem Cells Direct Axon Guidance via their Radial Fiber Scaffold

To determine the genes regulated by Fezf2 during neocortical development, we performed RNA-seq on Fezf2 +/+; Emx1-cre (Fezf2 wildtype) and Fezf2 fl/+; Emx1-Cre (Fezf2 Knockout) neocortices isolated from the P0 mice. After RNA isolation from the dissected tissue the samples were processed for library preperation using Illumina Poly-A RNA kit. Fezf2 can reprogram the upper layer neocortical neurons to form corticospinal tract. To understand the key transcriptomic changes driven by Fezf2 that drive this reprogramming, we performed in RNA-seq on the cells over-expressing Fezf2 and Gfp or Gfp. At E15.5, in utero electroporations targeting cortical plate, were performed in pregnant mice with Fezf2 and Gfp plasmids or with Gfp plasmid alone, which was used as control. At P3, the Gfp positive cells were FAC sorted from Fezf2 and Gfp, or Gfp cortices and processed for library preparations using NUGEN Ovation® V2 system. Libraries were sequenced using Illumina 2000 platform to generate 75bp single reads. At least 10 million uniquely mapped reads were obtained for each sample. To determine the genes directly regulated by FEZF2, we performed the ChIP-Seq on N2A cells that were transfected to over express FEZF2 using pCAG- Fezf2-V5 and pCAG-V5. After 48 hr, of transfection, cells were fixed and processed for ChIP seq. Libraries were prepared using Illumina TruSeq ChIP Libary Preparation Kit. Overall design: Determining the unique genes regulated by Fezf2 during the neocortex development by RNA-seq and ChIP-seq ChIP antibody information: V5 Tag Monoclonal Antibody (ThermoFisher Scientific, Cat# R960-25, RRID:AB_2556564)