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Single-cell RNA-sequencing analysis is one of the most effective tools for understanding specific cellular states. The use of single cells or pooled cells in RNA-seq analysis requires the isolation of cells from a tissue or culture. Although trypsin or more recently cold-active protease (CAP) has been used for cell dissociation, the extent to which the gene expression changes are suppressed has not been clarified. To this end, we conducted detailed profiling of the enzyme-dependent gene expression changes in mouse skeletal muscle progenitor cells, focusing on the enzyme treatment time, amount, and temperature. We found that the genes whose expression was changed by the enzyme treatment could be classified in a time-dependent manner, and that there were genes whose expression was changed independently of the enzyme treatment time, amount, and temperature. This study will be useful as reference data for genes that should be excluded or considered for RNA-seq analysis using enzyme isolation methods.