Single-cell transcriptomics of enriched rare neural lineages of Tribolium castaneum

Background Single-cell transcriptomic atlases have transformed our understanding of cell diversity. However, studies designed to profile entire tissues or organisms often omit or underrepresent rare cell populations. Enriching selected populations before sequencing offers a powerful approach to overcoming this limitation, but its application in insects has largely focused on Drosophila, limiting opportunities for comparative studies. Here, we establish complementary workflows for targeted transcriptomic profiling of rare neural populations in the red flour beetle, Tribolium castaneum. Results We developed two workflows combining population enrichment with single-cell or single-nucleus RNA sequencing. First, we generated cell suspensions from a transgenic reporter line labelling embryonic neuroblasts, enriched fluorescently labelled cells by fluorescence-activated cell sorting, and performed single-cell RNA sequencing. Second, we immunolabelled first-instar larval brains with an antibody against the nuclear transcription factor Rx, isolated and sorted labelled nuclei, and performed single-nucleus RNA sequencing. Both workflows yielded material suitable for transcriptomic profiling, demonstrating complementary reporter-based and antibody-based strategies for accessing selected neural populations at different developmental stages. Conclusions These workflows establish a methodological framework for targeted single-cell and single-nucleus transcriptomic studies of rare or specific neural populations in Tribolium. They provide practical starting points for adaptation to other insect systems, expanding opportunities for comparative investigations of neural development and cell-type evolution.