Microfluidic single-cell whole-transcriptome sequencing
Microfluidic single-cell whole-transcriptome sequencing
复制标题
微流控单细胞全转录组测序
DOI：
10.1073/pnas.1402030111
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发表时间：
2014-05-13
影响因子：
11.1
通讯作者：
Huang, Yanyi
中科院分区：
文献类型：
--
作者：
Streets, Aaron M.;Zhang, Xiannian;Huang, Yanyi
意义单个细胞的RNA测序能够测量不同细胞群体中的生物变异，并剖析隐藏在基因表达总体测量中的转录组复杂性。然而，单个细胞中的RNA含量低，阻碍了有效和一致的逆转录和扩增，限制了准确性，并掩盖了高技术噪声的生物变异。我们开发了一种微流控方法来从单个细胞中制备用于高通量转录组测序的cDNA.微流控平台促进了单细胞操作，最大限度地减少了污染，此外，还提高了检测灵敏度和测量精度，这是区分生物变异性和技术噪声所必需的。单细胞全转录组分析是量化细胞群体中基因表达异质性的有力工具。因此，最近已经开发了许多技术来对单个细胞进行转录组测序(RNA-Seq)。为了探测RNA含量有限的样本之间的微妙生物差异，仍然需要更精确和更灵敏的方法。我们采用了先前开发的单细胞RNA-Seq策略，该策略已显示出优异的灵敏度，并在单细胞全转录组分析的微流控平台中实现了化学。在这种方法中，单个细胞被捕获并在微流控设备中裂解，其中带有聚(A)尾巴的mRNAs被逆转录为cDNAs。然后使用下一代测序平台收集双链cDNA并进行测序。我们制备了94个文库，其中包括单个小鼠胚胎细胞和提取的RNA的技术复制，并对这项技术的性能进行了全面的表征。与基于试管的方法相比，微流控技术的实施提高了信使核糖核酸的检测灵敏度和测量精度。在每个细胞0.2万次读数的情况下，我们能够用10个单细胞重建大部分转录组。我们还量化了不同类型的小鼠胚胎细胞之间和内部的差异，发现提高的测量精度、检测灵敏度和实验吞吐量有助于区分生物可变性和技术噪声。通过这项工作，我们验证了单细胞RNA-Seq早期方法的优势，并表明将微流控技术与高通量测序相结合的好处将对单细胞转录组分析的大规模努力具有价值。
Significance RNA sequencing of single cells enables measurement of biological variation in heterogeneous cellular populations and dissection of transcriptome complexity that is masked in ensemble measurements of gene expression. The low quantity of RNA in a single cell, however, hinders efficient and consistent reverse transcription and amplification of cDNA, limiting accuracy and obscuring biological variation with high technical noise. We developed a microfluidic approach to prepare cDNA from single cells for high-throughput transcriptome sequencing. The microfluidic platform facilitates single-cell manipulation, minimizes contamination, and furthermore, provides improved detection sensitivity and measurement precision, which is necessary for differentiating biological variability from technical noise. Single-cell whole-transcriptome analysis is a powerful tool for quantifying gene expression heterogeneity in populations of cells. Many techniques have, thus, been recently developed to perform transcriptome sequencing (RNA-Seq) on individual cells. To probe subtle biological variation between samples with limiting amounts of RNA, more precise and sensitive methods are still required. We adapted a previously developed strategy for single-cell RNA-Seq that has shown promise for superior sensitivity and implemented the chemistry in a microfluidic platform for single-cell whole-transcriptome analysis. In this approach, single cells are captured and lysed in a microfluidic device, where mRNAs with poly(A) tails are reverse-transcribed into cDNA. Double-stranded cDNA is then collected and sequenced using a next generation sequencing platform. We prepared 94 libraries consisting of single mouse embryonic cells and technical replicates of extracted RNA and thoroughly characterized the performance of this technology. Microfluidic implementation increased mRNA detection sensitivity as well as improved measurement precision compared with tube-based protocols. With 0.2 M reads per cell, we were able to reconstruct a majority of the bulk transcriptome with 10 single cells. We also quantified variation between and within different types of mouse embryonic cells and found that enhanced measurement precision, detection sensitivity, and experimental throughput aided the distinction between biological variability and technical noise. With this work, we validated the advantages of an early approach to single-cell RNA-Seq and showed that the benefits of combining microfluidic technology with high-throughput sequencing will be valuable for large-scale efforts in single-cell transcriptome analysis.