Single-cell barcoding and sequencing using droplet microfluidics
Single-cell barcoding and sequencing using droplet microfluidics
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DOI：
10.1038/nprot.2016.154
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发表时间：
2017-01-01
期刊：
影响因子：
14.8
通讯作者：
Mazutis, Linas
中科院分区：
文献类型：
--
作者：
Zilionis, Rapolas;Nainys, Juozas;Mazutis, Linas
近年来，单细胞RNA测序已成为研究疾病和健康组织中细胞异质性的有力工具，但需要高通量的方法来获取无偏倚的细胞多样性。液滴微流控技术是捕获和处理数以千计的单个细胞，以大规模并行的方式进行全转录组或基因组分析的最有前途的候选者之一，使用的试剂最少。我们最近建立了一种名为In Drops的方法，它能够在一小时内索引15,000个细胞。细胞悬浮液首先用带有条形码DNA引物的水凝胶珠(HBs)包裹到纳升液滴中。然后，细胞被裂解，并通过逆转录(RT)反应对mRNA进行条形码(索引)。在这里，我们提供了用于(I)建立In Drops平台(1d)；(Ii)执行水凝胶微珠合成(4d)；(Iii)包裹和条形码细胞(1d)；以及(Iv)RNA-seq文库制备(2d)的细节。InDrops是一个强大且可扩展的平台，它的独特之处在于它能够以数千或数万个细胞的规模，从非常小的样本中捕获和分析75%的细胞。
Single-cell RNA sequencing has recently emerged as a powerful tool for mapping cellular heterogeneity in diseased and healthy tissues, yet high-throughput methods are needed for capturing the unbiased diversity of cells. Droplet microfluidics is among the most promising candidates for capturing and processing thousands of individual cells for whole-transcriptome or genomic analysis in a massively parallel manner with minimal reagent use. We recently established a method called in Drops, which has the capability to index > 15,000 cells in an hour. A suspension of cells is first encapsulated into nanoliter droplets with hydrogel beads (HBs) bearing barcoding DNA primers. Cells are then lysed and mRNA is barcoded (indexed) by a reverse transcription (RT) reaction. Here we provide details for (i) establishing an in Drops platform (1 d); (ii) performing hydrogel bead synthesis (4 d); (iii) encapsulating and barcoding cells (1 d); and (iv) RNA-seq library preparation (2 d). inDrops is a robust and scalable platform, and it is unique in its ability to capture and profile > 75% of cells in even very small samples, on a scale of thousands or tens of thousands of cells.