High-density spatial transcriptomics arrays for in situ tissue profiling

Tissue function relies on the precise spatial organization of cells characterized by distinct molecular profiles. Single-cell RNA-Seq captures molecular profiles but not spatial organization. Conversely, spatial profiling assays to date have lacked global transcriptome information, throughput or single-cell resolution. Here, we develop High-Density Spatial Transcriptomics (HDST), a method for RNA-Seq at high spatial resolution. Spatially barcoded reverse transcription oligonucleotides are coupled to beads that are randomly deposited into tightly packed individual microsized wells on a slide. The position of each bead is decoded with sequential hybridization using complementary oligonucleotides providing a unique bead-specific spatial address. We then capture, and spatially in situ barcode, RNA from the histological tissue sections placed on the HDST array. HDST recovers hundreds of thousands of transcript-coupled spatial barcodes per experiment at 2 um resolution. We demonstrate HDST in the mouse brain, use it to resolve spatial expression patterns and cell types, and show how to combine it with histological stains to relate expression patterns to tissue architecture and anatomy. HDST opens the way to spatial analysis of tissues at high resolution. Three sections of the main olfactory bulb from adult C57BL/6J mice组织功能依赖于具有独特分子特征的细胞的精准空间排布。单细胞RNA测序（Single-cell RNA-Seq）可捕获细胞的分子特征，但无法获取其空间排布信息。相较而言，迄今已报道的空间转录组分析技术均无法同时兼顾全局转录组信息、测序通量与单细胞分辨率。本研究开发了高密度空间转录组学（High-Density Spatial Transcriptomics, HDST），一种可实现高空间分辨率RNA测序的技术。将带有空间条形码的逆转录寡核苷酸偶联至微球，这些微球被随机沉积在载玻片上紧密排布的微型微孔中。通过使用携带独特微球特异性空间标识的互补寡核苷酸进行连续杂交，可解码每个微球的位置信息。随后，将组织学切片置于HDST芯片上，即可捕获其中的RNA并完成空间原位条形码标记。单次实验中，HDST可在2微米分辨率下获取数十万条与转录本关联的空间条形码。本研究在小鼠脑中验证了HDST的性能：利用该技术可解析空间表达模式与细胞类型，并展示了如何将其与组织学染色结合，以将表达模式与组织架构及解剖结构关联起来。HDST为高分辨率组织空间分析开辟了全新路径。本研究使用的实验样本为取自成年C57BL/6J小鼠主嗅球的三个组织切片。