Establishment and validation of the NEX-RiboTag system for profiling the excitatory neuronal translatome in the postnatal mouse forebrain

Excitatory neurons are the principal neurons of the mammalian cortex and hippocampus and are essential for postnatal circuit maturation. Although single-cell RNA sequencing has refined their molecular taxonomy, dissociation-induced stress artifacts and the disconnect between transcript abundance and translational output can limit functional interpretation. Bulk proteomics lacks cell-type specificity, while single-cell proteomics remains constrained by limited sensitivity and throughput. These limitations leave a gap between transcriptional identity and cell-type-resolved translational output. Because translation directly governs the selective recruitment of mRNAs for protein synthesis, defining excitatory neuron-specific translatome in vivo is valuable to bridge this gap. Here, we established and validated a NEX-RiboTag mouse line for targeted profiling of ribosome-associated mRNAs in cortical and hippocampal excitatory neurons. By crossing Neurod6 (NEX)-Cre mice with RiboTag reporter mice, we achieved Cre-dependent ribosomal tagging in excitatory neurons of the cortex and the hippocampus. RNA sequencing analysis at the 1-week postnatal stage demonstrated enrichment of excitatory neuronal markers and depletion of inhibitory neuronal and glial transcripts. Comparative analysis revealed a clear separation between the whole-tissue transcriptome and the ribosome-associated fractions, with enrichment of synaptic and metabolic pathways characteristic of excitatory neurons. Together, these datasets provide a valuable resource for investigating translational regulation in postnatal excitatory neurons and for studying molecular programs underlying neuronal maturation and synapse formation.