Low-input, deterministic profiling of single-cell transcriptomes reveals individual intestinal organoid subtypes comprised of single, dominant cell types [C1-HEK]

High-throughput single-cell RNA-sequencing (scRNA-seq) has transformed our ability to resolve cellular properties across systems. A key scRNA-seq catalyzer was the introduction of microdroplet-based systems, which vastly improved sample handling and cell throughput. While powerful, the current microfluidic systems are limited to high cell density (>1000 cells) samples. This prevents the efficient processing of individual, small tissues or rare cells, leading to respectively confounded mosaic cell population read-outs or failed capture of diagnostically interesting cells. In this study, we developed a deterministic, mRNA-capture bead and cell co-encapsulation droplet system, DisCo, that overcomes these limitations by enabling precise particle position and droplet sorting control through combined machine-vision and multilayer microfluidics. We demonstrate that DisCo is capable of processing samples containing few cells (< 100 cells) at high efficiencies( >70%). To underscore the unique capabilities of DisCo, we mapped the developmental process of 31 individual intestinal organoids at the single cell level. This uncovered extensive cellular heterogeneity among organoids, revealing two so far uncharacterized organoid subtypes, “gobloids” and spheroids, predominantly consisting of respectively Muc2+ goblet and Ly6a+ stem cells. Further Disco data analysis thereby revealed strongly increased Yap1 target gene expression in these spheroids, suggesting mechano sensing as the underlying mechanism for their spontaneous formation. Together, our novel “no-cell-left-behind” platform enables the deterministic processing of input cells, allowing high-resolution snapshots of cellular heterogeneity among rare cells or individual, small tissues or organoids.Together, our novel “no-cell-left-behind” platform enables the deterministic processing of input cells, allowing high-resolution snapshots of cellular heterogeneity among rare cells or individual, small tissues or organoids. To benchmark the cell recovery efficiency of the Fluidigm C1 platform, HEK 293T (ATCC Cat. No. SD-3515) cells were diluted to reach approximately 10, 20, 40 cells per µL.12 µL of cell solution was mixed with 8 µL of Suspension Reagent from Fluidigm. The obtained suspensions were generated separately from the same stock and then quantified in triplicate using microscopy by examining a volume of 2 x 2.5 µL of the suspension between two coverslips. Counts of all triplicates were averaged to estimate the cell input for 5 µL of Cell Mix to the C1 IFC. The experiment on the C1 machine was performed according to the kit’s manual: “SMART-Seq v4 Ultra Low Input RNA Kit for the Fluidigm C1 System, IFCs User Manual” (Clontech Laboratories, Inc.) using 10 – 17 µm 96-trap trap C1 IFC OpenApp chips. The prrotocol was run on SMART-Seq v4 (1861x/1862x/1863x) programs on the C1 machine. To verify successful loading and cell trapping, a Cell xCellence (Olympus) microscope was used. Final cDNA was quantified using the PicroGreen dsDNA assay and then tagmented using a Nextera XT kit.

高通量单细胞RNA测序（scRNA-seq）革新了我们解析跨系统细胞特性的能力。该领域的关键推动因素之一是基于微滴的系统的问世，其极大优化了样本处理流程并提升了细胞通量。尽管此类系统功能强大，但当前的微流控系统仅能处理高细胞密度（>1000个细胞）的样本，无法高效处理单个小型组织或稀有细胞样本，进而分别导致嵌合细胞群体测序结果混杂，或是无法捕获具有诊断价值的目标细胞。

本研究中，我们开发了一款基于确定性控制的mRNA捕获微球-细胞共包裹微滴系统DisCo，该系统结合机器视觉与多层微流控技术，可实现精准的粒子定位与微滴分选，从而克服了上述局限。实验证明，DisCo能够以高于70%的高效率处理仅含少量细胞（<100个细胞）的样本。

为凸显DisCo的独特性能，我们在单细胞层面解析了31个单个肠道类器官（organoids）的发育过程。该分析揭示了类器官间广泛存在的细胞异质性，并发现了两种此前未被表征的类器官亚型："gobloids"与球状类器官（spheroids），前者主要由Muc2+杯状细胞构成，后者则以Ly6a+干细胞为主。

进一步的DisCo数据分析显示，此类球状类器官中Yap1靶基因的表达水平显著上调，提示机械感知是其自发形成的潜在机制。

综上，这款全新的"no-cell-left-behind"平台可实现输入细胞的确定性处理，能够为稀有细胞、单个小型组织或类器官的细胞异质性提供高分辨率的分析快照。综上，这款全新的"no-cell-left-behind"平台可实现输入细胞的确定性处理，能够为稀有细胞、单个小型组织或类器官的细胞异质性提供高分辨率的分析快照。

为验证Fluidigm C1平台的细胞回收效率，我们将HEK 293T细胞（ATCC，货号SD-3515）稀释至每微升约10、20、40个细胞。取12微升细胞悬液与8微升Fluidigm公司的悬浮试剂混合。所得悬液均来自同一母液并分别制备，随后通过显微镜进行三次重复定量：在两张盖玻片之间取2×2.5微升的悬液进行计数。对三次重复的计数结果取平均值，以估算用于加载至C1集成流体回路（Integrated Fluidic Circuit，IFC）的5微升细胞混合液中的细胞投入量。本实验在C1仪器上按照试剂盒手册《Fluidigm C1系统适配集成流体回路用户手册：SMART-Seq v4超低起始量RNA试剂盒》（Clontech实验室公司）进行，使用的是10–17 µm规格的96阱C1 IFC OpenApp芯片。实验流程基于C1仪器上的SMART-Seq v4（1861x/1862x/1863x）程序运行。为验证样本加载与细胞捕获是否成功，我们使用了Cell xCellence（奥林巴斯）显微镜。最终的互补脱氧核糖核酸（cDNA）通过PicroGreen双链DNA（dsDNA）定量检测试剂盒进行定量，随后使用Nextera XT文库制备试剂盒进行片段化标记。