Single-cell RNA sequencing provides a deep understanding of gene expression patterns at the single-cell level, enabling researchers to study cellular heterogeneity and differentiation. Spatial transcriptomics, meanwhile, delivers spatial visualization and quantitative analysis of tissue sections. Here, we introduce a protocol to isolate single cells and obtain high-quality spatial transcriptomics data from bladder Ewing sarcoma tissue. We outline steps for sample collection, single-cell RNA sequencing, and spatial transcriptomics sequencing. For complete details on the use and execution of this protocol, please refer to Mao et al.