Well-ST-seq: cost-effective and cellular level spatial transcriptomics with high RNA capture efficiency

Spatial transcriptomic technologies are promising tools to reveal fine anatomical profiles of tissues. In the case of methodologies utilizing barcoded probe arrays, achieving a balance among probe barcoding complexity, cost, gene capture sensitivity, and spatial resolution is crucial for accelerating the spreading speed of spatial transcriptomic in basic science and clinical work. Here, we developed spatially cellular-level RNA-capture probe arrays using miniaturized microfluidic and microarray technologies. By leveraging the predetermined and cost-effective probe fixation characteristics of this methodology, we significantly reduced the consumable cost of the probe array to $0.31/mm2 and fabrication time to approximately 2 hours. Furthermore, the modification of the RNA-capture probe on sequencing slides by microfluidic chip does not rely on large imaging or printing instruments. Notably, the efficiency of the transcript captured by the probe array is even comparable to conventional single-cell RNA sequencing. Based on this technology, the stacked three-dimensional transcriptome atlas and the spatial cell heterogeneity of mouse brains were successfully visualized. Taken together, we present an experimental and analytical framework for the spatial investigation of mouse brain structures and cell phenotypes. Applying new approach Well-ST-seq for spatial transcriptomes sequencing to mouse tissues in different resolution (10 m, 20 m and 30m).空间转录组技术（Spatial transcriptomic technologies）是揭示组织精细解剖特征的极具应用前景的工具。在采用条形码探针阵列（barcoded probe arrays）的技术路线中，平衡探针条形码复杂度、成本、基因捕获灵敏度与空间分辨率，对于推动空间转录组技术在基础科学与临床工作中的普及至关重要。本研究基于微型化微流控与微阵列技术，开发了空间单细胞级RNA捕获探针阵列。借助该技术路线预先定制且兼具成本效益的探针固定特性，我们将探针阵列的耗材成本降至0.31美元/mm²，制备时长缩短至约2小时。此外，通过微流控芯片（microfluidic chip）对测序玻片上的RNA捕获探针进行修饰，无需依赖大型成像或打印设备。值得注意的是，该探针阵列捕获转录本的效率甚至可与传统单细胞RNA测序（conventional single-cell RNA sequencing）相媲美。基于该技术，我们成功可视化了小鼠大脑的堆叠式三维转录组图谱与空间细胞异质性。综上，本研究构建了用于小鼠大脑结构与细胞表型空间研究的实验与分析框架，并将新型方法Well-ST-seq应用于不同分辨率（10μm、20μm与30μm）的小鼠组织空间转录组测序。