A molecularly defined and spatially resolved cell atlas of the whole mouse brain [Bulk RNA sequencing]

In mammalian brains, millions to billions of cells form complex interaction networks to enable a wide range of functions. The enormous diversity and intricate organization of cells have impeded our understanding of the molecular and cellular basis of brain function. Recent advances in spatially resolved single-cell transcriptomics have enabled systematic mapping of the spatial organization of molecularly defined cell types in complex tissues. However, these approaches have only been applied to a few brain regions and a comprehensive cell atlas of the whole brain is still missing. Here, we imaged a panel of >1,100 genes in ~10 million cells across the entire adult mouse brain using multiplexed error-robust fluorescence in situ hybridization (MERFISH) and performed spatially resolved, single-cell expression profiling at the whole-transcriptome scale by integrating MERFISH and single-cell RNA-sequencing (scRNA-seq) data. Using this approach, we generated a comprehensive cell atlas of >5,000 transcriptionally distinct cell clusters, belonging to >300 major cell types, in the whole mouse brain with high molecular and spatial resolution. Registration of this atlas to the mouse brain common coordinate framework (CCF) allowed systematic quantifications of the cell-type composition and organization in individual brain regions. We further identified spatial modules characterized by distinct cell-type compositions and spatial gradients featuring gradual changes of cells. Finally, this high-resolution spatial map of cells, each with a transcriptome-wide expression profile, allowed us to infer cell-type-specific interactions between several hundred cell-type pairs and predict molecular (ligand-receptor) basis and functional implications of these cell-cell interactions. These results provide rich insights into the molecular and cellular architecture of the brain and a foundation for future functional investigations of neural circuits and their dysfunction in diseases. RNA expression levels of individual genes from three different whole mouse brains aged 56-62 days were extracted using RNAqueous Micro total RNA isolation kit. RNA quality was assessed using Agilent TapeStation and samples with an RNA integrity score >8 were kept for sequencing. RNA sequencing libraries were constructed using the Kapa mRNA HyperPrep Kits and were sequenced using the Illumina NextSeq500 platform performed by the Bauer Center Sequencing Core at Harvard University.在哺乳动物大脑中，数以百万乃至数十亿计的细胞构成复杂的相互作用网络，以支撑多种多样的大脑功能。细胞的巨大多样性与精细复杂的组织形式，长期阻碍了我们对大脑功能的分子与细胞基础的认知。近年来，空间分辨单细胞转录组学（spatially resolved single-cell transcriptomics）领域的进展，使得我们能够系统绘制复杂组织中经分子定义的细胞类型的空间排布图谱。然而，此类方法目前仅应用于少数脑区，完整覆盖全脑的综合细胞图谱仍有待构建。本研究中，我们借助多重误差鲁棒荧光原位杂交（multiplexed error-robust fluorescence in situ hybridization, MERFISH）技术，对成年小鼠全脑约1000万个细胞中的超过1100个基因进行成像；并通过整合MERFISH与单细胞RNA测序（single-cell RNA-sequencing, scRNA-seq）数据，实现了全转录组层面的空间分辨单细胞表达谱分析。借助该方法，我们成功构建了兼具高分子分辨率与空间分辨率的成年小鼠全脑综合细胞图谱，涵盖超过5000个转录特征迥异的细胞簇，归属于300余种主要细胞类型。将该图谱配准至小鼠脑通用坐标框架（common coordinate framework, CCF）后，我们能够系统量化各个脑区的细胞类型组成与排布模式。我们进一步鉴定出以独特细胞类型组成为特征的空间模块，以及呈现细胞渐进性变化的空间梯度分布。最终，这份具备高分辨率的细胞空间图谱（每个细胞均带有全转录组表达谱），使我们能够推断数百对细胞类型间的细胞类型特异性相互作用，并预测这些细胞间相互作用的分子（配体-受体）基础及其功能意义。本研究结果为解析大脑的分子与细胞架构提供了丰富的认知基础，同时也为后续神经环路功能研究及其疾病相关功能异常的探索奠定了重要框架。 本研究针对3只56至62日龄的成年小鼠全脑，采用RNAqueous Micro总RNA提取试剂盒（RNAqueous Micro total RNA isolation kit）提取各基因的RNA表达水平；使用安捷伦TapeStation（Agilent TapeStation）评估RNA质量，保留RNA完整性评分＞8的样本用于测序。测序文库构建采用Kapa mRNA HyperPrep试剂盒（Kapa mRNA HyperPrep Kits），测序工作由哈佛大学鲍尔中心测序核心实验室（Bauer Center Sequencing Core at Harvard University）依托Illumina NextSeq500平台完成。