Loss Of Neural Stem Cell O-Glcnacylation Regulates An Age-Related Fate Switch In The Mature Hippocampus

To gain mechanistic insight into the role of O-GlcNAc in adult hippocampal neural stem cells (NSCs), we treated primary NSCs with a pharmacological inhibitor of Ogt or a lentivirus encoding an shRNA construct targeting Ogt. We determined that loss of O-GlcNAc in NSCs impairs proliferation and activates programs associated with glial differentiation. Overall design: mRNA of NSCs was isolated by lysis with TRIzol Reagent (Thermo Fisher Scientific), separation with chloroform (0.2mL per mL TRIzol), and precipitated with isopropyl alcohol according to manufacturer's instructions. RNA-Seq libraries were constructed using the Smart-Seq2 protocol from Trombetta et al. 2014. Final libraries were purified with Ampure beads and quantified using a qPCR Library Quantification Kit (Kapa Biosystems). Libraries were pooled for sequencing on an Illumina HiSeq 2500 (paired reads 2x100bp). Alignment of RNA-sequencing reads to the transcriptome was performed using STAR with ENCODE standard options, read counts were generated using rsem, and differential expression analysis was performed in R using DESeq2 package (Love et al., 2014). All bioinformatics analyses were performed on Sherlock, a Stanford HPC cluster.