Single-cell RNA-sequencing (scRNA-seq) has transformed our ability to resolve cellular properties across systems. However, current scRNA-seq platforms are one-size-fits-all approaches that are tailored toward large cell inputs (> 1,000 cells), rendering them inefficient and costly when processing small, individual tissue samples. This important drawback tends to be resolved by loading bulk samples, but this yields confounded mosaic cell population read-outs. To overcome these technological limitations, we developed a deterministic, mRNA-capture bead and cell co-encapsulation dropleting system, DisCo. We demonstrate that DisCo enables precise particle and cell positioning and droplet sorting control through combined machine-vision and multilayer microfluidics. In comparison to other microfluidics systems, the active flow control driving DisCo, enables continuous operation and processing of low-input samples (< 100 cells) at high capture efficiency (> 70%). To underscore the unique capabilities of our approach, we analyzed intestinal organoid development by “DisCo-ing” 31 individual organoids at varying developmental stages. This revealed extensive organoid heterogeneity, identifying distinct subtypes including a regenerative fetal-like Ly6a⁺ stem cell population which persists as symmetrical cysts even under differentiation conditions. Furthermore, we uncovered a so far uncharacterized “gobloid” subtype consisting predominantly of precursor and mature (Muc2⁺) goblet cells. These findings demonstrate the unique power of DisCo in providing high-resolution snapshots of cellular heterogeneity among small, individual tissues.

中文翻译：

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单个肠类器官的确定性scRNA-seq揭示了新的亚型和不同的干细胞库共存

单细胞RNA测序（scRNA-seq）改变了我们解析整个系统细胞特性的能力。但是，当前的scRNA-seq平台是一种“一刀切”的方法，专门针对大型细胞输入（> 1,000个细胞）而设计，在处理小型单个组织样本时，它们效率低下且成本高昂。这个重要的缺点往往可以通过装载大量样品来解决，但是这会混淆马赛克细胞群的读数。为了克服这些技术限制，我们开发了一个d etermin是抽动症，基因捕获珠和细胞合作-胶囊滴注系统DisCo。我们展示了DisCo通过结合机器视觉和多层微流体技术实现精确的粒子和细胞定位以及液滴分类控制。与其他微流控系统相比，主动流量控制驱动DisCo能够以高捕获效率（> 70％）连续运行和处理低输入量样品（<100个细胞）。为了强调我们方法的独特功能，我们通过“ DisCo-ing” 31个在不同发育阶段的个体类器官来分析肠道类器官的发育。这揭示了广泛的类器官异质性，确定了不同的亚型，包括可再生的胎儿样Ly6a ⁺干细胞群体，即使在分化条件下也能以对称囊肿的形式存在。此外，我们发现了迄今为止尚未鉴定的“球状”亚型，主要由前体和成熟（Muc 2 ⁺）杯状细胞组成。这些发现证明了DisCo在提供单个个体组织间细胞异质性的高分辨率快照方面的独特能力。