Objective To compare the digestive efficiency of collagenase Ⅳ and trypsin on renal tissue at different time points and explore the applicability of magnetic bead separation for single cell sequencing.Methods The renal tissues were digested with col⁃ lagenase Ⅳ and trypsin for 15 min，30 min and 15 min+15 min，respectively.The digestive efficiency of the two enzymes on renal tis⁃ sue at different time points were compared，and the best digestion method was selected.CD45+ cells were separated from the renal cell suspension using magnetic beads.The number，activity and clumping rate of isolated cells were detected，and cell status was evaluated according to single cell sequencing results.Results The renal tissue was almost completely digested after 30 min of enzyme treatment.Under the same digestion time and method，the digestive efficiency of collagenase Ⅳ was obviously better than that of trypsin.The 15 min+15 min digestion method resulted in fewer cell fragments and a cleaner background compared to the 30 min digestion method.CD45+ cells isolated from the cell suspension obtained by the 15 min+15 min digestion with collagenase IV met the requirements for single-cell sequencing，including the total number of cells，survival rate，and agglomeration rate.Single cell sequencing results showed that all cells expressed CD45+，and the proportion of mitochondrial gene expression was low，indicating that the cells were less stimu⁃ lated by external factors.Conclusion Compared to trypsin，collagenase IV had a better digestive efficiency on renal tissue under the same digestion time，which reduced the production of cell fragments.The CD45+ renal single-cell suspension obtained by magnetic bead separation met the requirements for single-cell sequencing and had high purity，which contributed to the accuracy of subsequent sequencing results analysis.