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Summary: Multiplexed single-cell RNA-sequencing (scRNA-seq) enables investigating several biological samples in one scRNA-seq experiment. Here, we use antibodies tagged with a hashtag oligonucleotide (Ab-HTO) to label each sample, and 10× Genomics technology to analyze single-cell gene expression. Advantages of sample multiplexing are to reduce the cost of scRNA-seq assay and to avoid batch effect. It may also facilitate cell-doublet removal and the merging of several scRNA-seq assays. Herein, we apply multiplexed scRNA-seq to investigate mouse thymocytes and splenic T lymphocytes development.For complete details on the use and execution of this protocol, please refer to Nozais et al. (2021).