Spatial transcriptome profiling by MERFISH reveals subcellular RNA compartmentalization and cell cycle-dependent gene expression
Spatial transcriptome profiling by MERFISH reveals subcellular RNA compartmentalization and cell cycle-dependent gene expression
复制标题
DOI：
10.1073/pnas.1912459116
复制
发表时间：
2019-09-24
影响因子：
11.1
通讯作者：
Zhuang, Xiaowei
中科院分区：
文献类型：
--
作者：
Xia, Chenglong;Fan, Jean;Zhuang, Xiaowei
rna的表达谱和空间分布调节着许多细胞功能。基于图像的转录组学方法为测量天然环境中单个细胞中rna的表达和空间信息提供了强大的手段。在这些方法中，多路错误鲁棒荧光原位杂交（MERFISH）通过大规模多路单分子FISH测量在转录组尺度上实现了空间分辨RNA定量。在这里，我们提高了MERFISH的基因通量，并展示了在单个细胞中同时测量近10,000个基因的RNA转录本，检测效率接近80%，误读率接近4%。我们结合MERFISH和细胞结构成像来确定rna的亚细胞区隔化。我们通过显示内质网分泌组转录物的富集来验证这种方法，并进一步揭示了长链非编码rna、保留内含子的rna和细胞核中蛋白质编码mrna亚群的富集。利用空间分辨RNA分析，我们开发了一种方法来确定RNA速度在原位使用核与细胞质RNA计数的平衡。我们应用这种方法来推断细胞的伪时间顺序，并鉴定了不同细胞周期状态的细胞，揭示了大约1600个基因，这些基因具有假定的细胞周期依赖性表达，并且随着细胞在细胞周期阶段的进展而逐渐发生转录谱变化。我们的分析进一步揭示了细胞周期依赖和细胞周期独立的转录不同细胞的空间异质性。我们设想，MERFISH能够以高检测效率和准确性执行空间分辨率，全基因组RNA分析，有助于解决从细胞中基因表达调控到细胞命运和组织发育的广泛问题。
The expression profiles and spatial distributions of RNAs regulate many cellular functions. Image-based transcriptomic approaches provide powerful means to measure both expression and spatial information of RNAs in individual cells within their native environment. Among these approaches, multiplexed error-robust fluorescence in situ hybridization (MERFISH) has achieved spatially resolved RNA quantification at transcriptome scale by massively multiplexing single-molecule FISH measurements. Here, we increased the gene throughput of MERFISH and demonstrated simultaneous measurements of RNA transcripts from similar to 10,000 genes in individual cells with similar to 80% detection efficiency and similar to 4% misidentification rate. We combined MERFISH with cellular structure imaging to determine subcellular compartmentalization of RNAs. We validated this approach by showing enrichment of secretome transcripts at the endoplasmic reticulum, and further revealed enrichment of long noncoding RNAs, RNAs with retained introns, and a subgroup of protein-coding mRNAs in the cell nucleus. Leveraging spatially resolved RNA profiling, we developed an approach to determine RNA velocity in situ using the balance of nuclear versus cytoplasmic RNA counts. We applied this approach to infer pseudotime ordering of cells and identified cells at different cell-cycle states, revealing similar to 1,600 genes with putative cell cycle-dependent expression and a gradual transcription profile change as cells progress through cell-cycle stages. Our analysis further revealed cell cycle-dependent and cell cycle-independent spatial heterogeneity of transcriptionally distinct cells. We envision that the ability to perform spatially resolved, genome-wide RNA profiling with high detection efficiency and accuracy by MERFISH could help address a wide array of questions ranging from the regulation of gene expression in cells to the development of cell fate and organization in tissues.