Transcriptome mapping in the 3D brain RNA sequencing samples the entire transcriptome but lacks anatomical information. In situ hybridization, on the other hand, can only profile a small number of transcripts. In situ sequencing technologies address these shortcomings but face a challenge in dense, complex tissue environments. Wang et al. combined an efficient sequencing approach with hydrogel-tissue chemistry to develop a multidisciplinary technology for three-dimensional (3D) intact-tissue RNA sequencing (see the Perspective by Knöpfel). More than 1000 genes were simultaneously mapped in sections of mouse brain at single-cell resolution to define cell types and circuit states and to reveal cell organization principles. Science, this issue p. eaat5691; see also p. 328 Wang et al. describe the development and application of an RNA sequencing technology to define cell types and circuit states in the mouse brain. INTRODUCTION Single-cell RNA sequencing has demonstrated that both stable cell types and transient cell states can be discovered and defined by transcriptomes. In situ transcriptomic methods can map both RNA quantity and position; however, it remains challenging to simultaneously satisfy key technological requirements such as efficiency, signal intensity, accuracy, scalability to large gene numbers, and applicability to three-dimensional (3D) volumes. Well-established single-molecule fluorescence in situ hybridization (FISH) approaches (such as MERFISH and seqFISH) have high detection efficiency but require long RNA species (more than 1000 nucelotides) and yield lower intensity than that of enzymatic amplification methods (tens versus thousands of fluorophores per RNA molecule). Other pioneering in situ sequencing methods (via padlock probes and fluorescent in situ sequencing) use enzymatic amplification, thus achieving high intensity but with room to improve on efficiency. RATIONALE We have developed, validated, and applied STARmap (spatially-resolved transcript amplicon readout mapping). STARmap begins with labeling of cellular RNAs by pairs of DNA probes followed by enzymatic amplification so as to produce a DNA nanoball (amplicon), which eliminates background caused by mislabeling of single probes. Tissue can then be transformed into a 3D hydrogel DNA chip by anchoring DNA amplicons via an in situ–synthesized polymer network and removing proteins and lipids. This form of hydrogel-tissue chemistry replots amplicons onto an optically transparent hydrogel coordinate system; then, to identify and quantify RNA species-abundance manifested by DNA amplicons, the identity of each species is encoded as a five-base barcode and read out by means of an in situ sequencing method that decodes DNA sequence in multicolor fluorescence. Using a new two-base sequencing scheme (SEDAL), STARmap was found to simultaneously detect more than 1000 genes over six imaging cycles, in which sequencing errors in any cycle cause misdecoding and are effectively rejected. RESULTS We began by (i) detecting and quantifying a focused 160-gene set (including cell type markers and activity-regulated genes) simultaneously in mouse primary visual cortex; (ii) clustering resulting per-cell gene expression patterns into a dozen distinct inhibitory, excitatory, and non-neuronal cell types; and (iii) mapping the spatial distribution of all of these cell types across layers of cortex. For validation, per-cell-type gene expression was found to correlate well both with in situ hybridization results and with single-cell RNA sequencing, and widespread up-regulation of activity-regulated genes was observed in response to visual stimulation. We next applied STARmap to a higher cognitive area (the medial prefrontal cortex) and discovered a more complex distribution of cell types. Last, we extended STARmap to much larger numbers of genes and spatial scales; we measured 1020 genes simultaneously in sections—obtaining results concordant with the 160-gene set—and measured 28 genes across millimeter-scale volumes encompassing ~30,000 cells, revealing 3D patterning principles that jointly characterize a broad and diverse spectrum of cell types. CONCLUSION STARmap combines hydrogel-tissue chemistry and in situ DNA sequencing to achieve intact-tissue single-cell measurement of expression of more than a thousand genes. In the future, combining this intact-system gene expression measurement with complementary cellular-resolution methodologies (with which STARmap is designed to be compatible)—including in vivo activity recording, optogenetic causal tests, and anatomical connectivity in the same cells—will help bridge molecular, cellular, and circuit scales of neuroscience. STARmap for 3D transcriptome imaging and molecular cell typing. STARmap is an in situ RNA-sequencing technology that transforms intact tissue into a 3D hydrogel-tissue hybrid and measures spatially resolved single-cell transcriptomes in situ. Error- and background-reduction mechanisms are implemented at multiple layers, enabling precise RNA quantification, spatially resolved cell typing, scalability to large gene numbers, and 3D mapping of tissue architecture. Retrieving high-content gene-expression information while retaining three-dimensional (3D) positional anatomy at cellular resolution has been difficult, limiting integrative understanding of structure and function in complex biological tissues. We developed and applied a technology for 3D intact-tissue RNA sequencing, termed STARmap (spatially-resolved transcript amplicon readout mapping), which integrates hydrogel-tissue chemistry, targeted signal amplification, and in situ sequencing. The capabilities of STARmap were tested by mapping 160 to 1020 genes simultaneously in sections of mouse brain at single-cell resolution with high efficiency, accuracy, and reproducibility. Moving to thick tissue blocks, we observed a molecularly defined gradient distribution of excitatory-neuron subtypes across cubic millimeter–scale volumes (>30,000 cells) and a short-range 3D self-clustering in many inhibitory-neuron subtypes that could be identified and described with 3D STARmap.

中文翻译：

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单细胞转录状态的三维完整组织测序

3D 脑 RNA 测序中的转录组映射对整个转录组进行采样，但缺乏解剖信息。另一方面，原位杂交只能描述少量的转录本。原位测序技术解决了这些缺点，但在密集、复杂的组织环境中面临挑战。王等。将有效的测序方法与水凝胶组织化学相结合，开发了一种用于三维 (3D) 完整组织 RNA 测序的多学科技术（参见 Knöpfel 的观点）。超过 1000 个基因以单细胞分辨率同时映射到小鼠大脑的各个部分，以定义细胞类型和电路状态并揭示细胞组织原理。科学，这个问题 p。eaat5691；另见 p. 328 王等人。描述 RNA 测序技术的开发和应用，以确定小鼠大脑中的细胞类型和电路状态。简介 单细胞 RNA 测序表明，稳定的细胞类型和瞬态细胞状态都可以通过转录组发现和定义。原位转录组学方法可以绘制 RNA 的数量和位置；然而，同时满足效率、信号强度、准确性、大基因数量的可扩展性和三维 (3D) 体积的适用性等关键技术要求仍然具有挑战性。完善的单分子荧光原位杂交 (FISH) 方法（例如 MERFISH 和 seqFISH）具有高检测效率，但需要长 RNA 种类（超过 1000 个核苷酸）并且产生的强度低于酶促扩增方法（数十对数千）每个 RNA 分子的荧光团）。其他开创性的原位测序方法（通过挂锁探针和荧光原位测序）使用酶促扩增，从而实现高强度，但仍有提高效率的空间。基本原理 我们开发、验证并应用了 STARmap（空间分辨的转录本扩增子读出映射）。STARmap 首先通过成对的 DNA 探针标记细胞 RNA，然后进行酶促扩增，从而产生 DNA 纳米球（扩增子），这消除了由单个探针错误标记引起的背景。然后，通过原位合成的聚合物网络锚定 DNA 扩增子并去除蛋白质和脂质，可以将组织转化为 3D 水凝胶 DNA 芯片。这种形式的水凝胶组织化学将扩增子重新绘制到光学透明的水凝胶坐标系上；然后，为了识别和量化 DNA 扩增子表现出的 RNA 物种丰度，每个物种的身份被编码为一个五碱基条形码，并通过原位测序方法读取多色荧光中的 DNA 序列。使用新的双碱基测序方案 (SEDAL)，STARmap 被发现在六个成像周期内同时检测 1000 多个基因，其中任何周期中的测序错误都会导致错误解码并被有效拒绝。结果 我们首先 (i) 在小鼠初级视觉皮层中同时检测和量化集中的 160 个基因组（包括细胞类型标记和活性调节基因）；(ii) 将产生的每个细胞基因表达模式聚类成十几种不同的抑制性、兴奋性和非神经元细胞类型；(iii) 绘制所有这些细胞类型在皮质层间的空间分布图。为了验证，发现每个细胞类型的基因表达与原位杂交结果和单细胞 RNA 测序都有很好的相关性，并且观察到响应视觉刺激的活性调节基因的广泛上调。接下来，我们将 STARmap 应用于更高的认知区域（内侧前额叶皮层），并发现了更复杂的细胞类型分布。最后的，我们将 STARmap 扩展到更多的基因和空间尺度；我们在切片中同时测量了 1020 个基因——获得与 160 个基因组一致的结果——并在包含约 30,000 个细胞的毫米级体积中测量了 28 个基因，揭示了 3D 模式原理，这些原理共同表征了广泛而多样的细胞类型。结论 STARmap 将水凝胶组织化学和原位 DNA 测序相结合，实现了对超过一千个基因表达的完整组织单细胞测量。将来，将这种完整系统的基因表达测量与互补的细胞分辨率方法（STARmap 旨在与之兼容）相结合——包括体内活动记录、光遗传学因果测试和同一细胞中的解剖连接——将有助于弥合分子，细胞，和神经科学的电路尺度。用于 3D 转录组成像和分子细胞分型的 STARmap。STARmap 是一种原位 RNA 测序技术，可将完整组织转化为 3D 水凝胶-组织混合体，并原位测量空间分辨的单细胞转录组。错误和背景减少机制在多个层次上实施，从而实现精确的 RNA 量化、空间分辨的细胞分型、大基因数量的可扩展性和组织结构的 3D 映射。在以细胞分辨率保留三维 (3D) 位置解剖结构的同时检索高含量基因表达信息一直很困难，限制了对复杂生物组织结构和功能的综合理解。我们开发并应用了一种 3D 完整组织 RNA 测序技术，称为 STARmap（空间分辨转录扩增子读出映射），它集成了水凝胶组织化学、靶向信号放大和原位测序。STARmap 的功能通过在小鼠大脑切片中以高效率、准确性和可重复性在单细胞分辨率下同时映射 160 到 1020 个基因来测试。转向厚组织块，我们观察到兴奋性神经元亚型在立方毫米尺度体积（>30,000 个细胞）中的分子定义梯度分布，以及许多可以识别和描述的抑制性神经元亚型中的短程 3D 自聚类与 3D 星图。STARmap 的功能通过在小鼠大脑切片中以高效率、准确性和可重复性在单细胞分辨率下同时映射 160 到 1020 个基因来测试。转向厚组织块，我们观察到兴奋性神经元亚型在立方毫米尺度体积（>30,000 个细胞）中的分子定义梯度分布，以及许多可以识别和描述的抑制性神经元亚型中的短程 3D 自聚类与 3D 星图。STARmap 的功能通过在小鼠大脑切片中以高效率、准确性和可重复性在单细胞分辨率下同时映射 160 到 1020 个基因来测试。转向厚组织块，我们观察到兴奋性神经元亚型在立方毫米尺度体积（>30,000 个细胞）中的分子定义梯度分布，以及许多可以识别和描述的抑制性神经元亚型中的短程 3D 自聚类与 3D 星图。