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Summary: Single-cell combinatorial indexing RNA sequencing (sci-RNA-seq3) enables high-throughput single-nucleus transcriptomic profiling of multiple samples in one experiment. Here, we describe an optimized protocol of mouse kidney nuclei isolation and sci-RNA-seq3 library preparation. The use of a dounce tissue homogenizer enables nuclei extraction with high yield. Fixed nuclei are processed for sci-RNA-seq3, and self-loaded transposome Tn5 is used for tagmentation in library generation. The step-by-step protocol allows researchers to generate scalable single-cell transcriptomic data with common laboratory supplies at low cost.For complete details on the use and execution of this protocol, please refer to Li et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.