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. 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.