High-throughput single-cell gene-expression profiling with multiplexed error-robust fluorescence in situ hybridization
High-throughput single-cell gene-expression profiling with multiplexed error-robust fluorescence in situ hybridization
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DOI：
10.1073/pnas.1612826113
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发表时间：
2016-09-27
影响因子：
11.1
通讯作者：
Zhuang, Xiaowei
中科院分区：
文献类型：
--
作者：
Moffitt, Jeffrey R.;Hao, Junjie;Zhuang, Xiaowei
基于图像的单细胞转录组学方法，即通过成像对 RNA 种类进行原位识别和计数，已成为基于分离细胞 RNA 测序的单细胞方法的有力补充。这些基于图像的方法自然地保留了细胞内 RNA 的天然空间背景和组织内细胞的组织，这对于解决许多生物学问题非常重要。然而，这些基于图像的方法的吞吐量相对较低。在这里，我们报告了导致多重抗误差荧光原位杂交 (MERFISH) 测量通量大幅增加的进展，MERFISH 是一种基于图像的单细胞转录组学方法。在 MERFISH 中，RNA 通过组合标记方法进行识别，该方法用防错条形码编码 RNA 种类，然后进行连续几轮单分子荧光原位杂交 (smFISH) 来读取这些条形码。在这里，我们通过组合改进将 MERFISH 的吞吐量提高了两个数量级，包括使用化学裂解代替光漂白来去除连续轮 smFISH 成像之间的荧光信号、增加成像视野以及使用多色成像。通过这些改进，我们对超过 100,000 个人类细胞进行了 RNA 分析，在单次 18 小时测量中测量了多达 40,000 个细胞。这种吞吐量应该会大大扩展 MERFISH 可以解决的生物学问题的范围。
Image-based approaches to single-cell transcriptomics, in which RNA species are identified and counted in situ via imaging, have emerged as a powerful complement to single-cell methods based on RNA sequencing of dissociated cells. These image-based approaches naturally preserve the native spatial context of RNAs within a cell and the organization of cells within tissue, which are important for addressing many biological questions. However, the throughput of these image-based approaches is relatively low. Here we report advances that lead to a drastic increase in the measurement throughput of multiplexed error-robust fluorescence in situ hybridization (MERFISH), an image-based approach to single-cell transcriptomics. In MERFISH, RNAs are identified via a combinatorial labeling approach that encodes RNA species with error-robust barcodes followed by sequential rounds of single-molecule fluorescence in situ hybridization (smFISH) to read out these barcodes. Here we increase the throughput of MERFISH by two orders of magnitude through a combination of improvements, including using chemical cleavage instead of photobleaching to remove fluorescent signals between consecutive rounds of smFISH imaging, increasing the imaging field of view, and using multicolor imaging. With these improvements, we performed RNA profiling in more than 100,000 human cells, with as many as 40,000 cells measured in a single 18-h measurement. This throughput should substantially extend the range of biological questions that can be addressed by MERFISH.