An optimized protocol for single cell transcriptional profiling by combinatorial indexing

Single cell combinatorial indexing RNA sequencing (sci-RNA-seq) is a powerful method for recovering gene expression data from an exponentially scalable number of individual cells or nuclei. However, sci-RNA-seq is a complex protocol that has historically exhibited variable performance on different tissues, as well as lower sensitivity than alternative methods. Here we report a simplified, optimized version of the three-level sci-RNA-seq protocol that is faster, higher yield, more robust, and more sensitive, than the original sci-RNA-seq3 protocol, with reagent costs on the order of 1 cent per cell or less. We showcase the optimized protocol via whole organism analysis of an E16.5 mouse embryo, profiling ~380,000 nuclei in a single experiment. Finally, we introduce a "tiny sci-*" protocol for experiments where input is extremely limited. Nuclei were isolated from one E16.5 mouse embryo to perform the optimized sci-RNA-seq3 protocol.单细胞组合索引RNA测序（Single cell combinatorial indexing RNA sequencing，sci-RNA-seq）是一种可从指数级扩展规模的单个细胞或细胞核中获取基因表达数据的高效技术。然而，sci-RNA-seq属于复杂的实验方案，既往在不同组织中常表现出性能波动，且灵敏度不及部分同类替代方法。本研究报道了一种简化优化后的三级sci-RNA-seq实验方案：相较于原始sci-RNA-seq3方案，该方案耗时更短、得率更高、稳定性更强且灵敏度更优，试剂成本可低至每细胞约1美分及以下。我们通过对E16.5小鼠胚胎开展全生物体分析验证了该优化方案，单次实验即可完成约38万个细胞核的测序分析。最后，我们针对输入样本极有限的实验场景，推出了“微型sci-*”实验方案。本研究从1枚E16.5小鼠胚胎中分离细胞核，用以开展优化后的sci-RNA-seq3实验。