Cell atlas projects and high-throughput perturbation screens require single-cell sequencing at a scale that is challenging with current technology. To enable cost-effective single-cell sequencing for millions of individual cells, we developed ‘single-cell combinatorial fluidic indexing’ (scifi). The scifi-RNA-seq assay combines one-step combinatorial preindexing of entire transcriptomes inside permeabilized cells with subsequent single-cell RNA-seq using microfluidics. Preindexing allows us to load several cells per droplet and computationally demultiplex their individual expression profiles. Thereby, scifi-RNA-seq massively increases the throughput of droplet-based single-cell RNA-seq, and provides a straightforward way of multiplexing thousands of samples in a single experiment. Compared with multiround combinatorial indexing, scifi-RNA-seq provides an easy and efficient workflow. Compared to cell hashing methods, which flag and discard droplets containing more than one cell, scifi-RNA-seq resolves and retains individual transcriptomes from overloaded droplets. We benchmarked scifi-RNA-seq on various human and mouse cell lines, validated it for primary human T cells and applied it in a highly multiplexed CRISPR screen with single-cell transcriptome readout of T cell receptor activation.

细胞图谱项目和高通量扰动筛选需要单细胞测序，其规模对当前技术来说具有挑战性。为了能够对数百万个单个细胞进行经济高效的单细胞测序，我们开发了“单细胞组合流体索引”（scifi）。scifi-RNA-seq 测定将透化细胞内整个转录组的一步组合预索引与随后使用微流体的单细胞 RNA-seq 相结合。预索引使我们能够在每个液滴中加载多个细胞，并通过计算对它们各自的表达谱进行解复用。因此，scifi-RNA-seq 极大地提高了基于液滴的单细胞 RNA-seq 的通量，并提供了一种在单个实验中多重分析数千个样本的简单方法。与多轮组合索引相比，scifi-RNA-seq 提供了简单高效的工作流程。与标记并丢弃包含多个细胞的液滴的细胞散列方法相比，scifi-RNA-seq 可以解析并保留来自超载液滴的单个转录组。我们在各种人类和小鼠细胞系上对 scifi-RNA-seq 进行了基准测试，对原代人类 T 细胞进行了验证，并将其应用于高度多重 CRISPR 筛选，并可读取 T 细胞受体激活的单细胞转录组读数。