Simultaneous profiling of native-state proteomes and transcriptomes of brain cell types using proximity labeling

Phenotyping cells at transcriptomic and proteomic levels is an essential step to understanding cellular contributions to development, aging, injury, and disease. Since proteome and transcrip-tome level abundances modestly correlate, complementary profiling of both is needed. We report a method called simultaneous protein and RNA -omics (SPARO) to capture the cell type-specific transcriptome and proteome simultaneously in vitro and in vivo. SPARO leverages TurboID to biotinylate RNA-interacting cytosolic proteins, enabling enrichment of proteins for proteomics and protein-associated RNA for transcriptomics. We validate SPARO first using well-controlled in vitro systems to verify that the proteomes and transcriptomes obtained reflect the global proteo-mes and transcriptomes. The effect of neuroinflammatory activation by lipopolysaccharide is al-so faithfully captured. We apply SPARO to obtain native-state proteomes and transcriptomes from astrocytes and neurons, thereby validating the approach in vivo. We interrogate mRNA-protein concordance and discordance, providing insights into molecular processes that exhibit uniform or cell type-specific patterns. Overall design: Cell type-specific transcriptomes of BV2-microglia and HEK293 cells in vitro and Aldh1l1-expressing astrocytes and Camk2a-expressing neurons in vivo were obtained using the proximity labeling enzyme, TurboID, that biotinylates proteins that interact with RNA (e.g., ribosomal and RNA-binding proteins). Following streptavidin or A/G bead affinity purification, RNA was extracted and sent for RNA-sequencing. HEK293 cells expressing TurboID with and without a nuclear export sequence were contrasted. The TurboID-enriched and RiboTag-enriched astrocyte in vivo transcriptomes were also compared. MicroRNA-sequencing from cortical Aldh1l1-expressing astrocytes and the bulk cortex in vivo was also completed.