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Single-cell or single-nucleus transcriptomics is a powerful tool for identifying cell types and cell states. However, hypotheses derived from these assays, including gene expression information, require validation, and their functional relevance needs to be established. The choice of validation depends on numerous factors. Here, we present types of orthogonal and functional validation experiment to strengthen preliminary findings obtained using single-cell and single-nucleus transcriptomics as well as the challenges and limitations of these approaches.