Simultaneous multiplexed amplicon sequencing and transcriptome profiling in single cells. Simultaneous multiplexed amplicon sequencing and transcriptome profiling in single cells

We developed a tunable ligation technology to alter beads used for to capture mRNA for single-cell RNA sequencing (e.g. Drop-seq); we call this technique droplet assisted RNA targeting by single cell sequencing, or DART-seq. In the study, we applied the new technology to capture viral genome transcripts of recombinant type 3 Dearing orthoreovirus infecting murine L929 cells. In addition to capture of targeted viral gene transcripts, we were also able to simultaneosly pull-down polyadenylated mRNA from the L929 cells to observe host-pathogen interactions. We also applied the technology to efficiently measure natively paired heavy and light chain amplicons from B lymphocyte cells in a collection of human peripheral blood mononuclear cells. Overall design: Commercial Drop-seq beads (Chemgene) were altered by a tunable ligation chemistry to capture targets of interest while maintaining the ability to capture polyadenylated mRNAs. Altered or normal beads were resuspended in lysis buffer and were coencapsulated with single cells (either infected murine L929 cells or, in another experiment, human PBMCs) using a microfluidic device (as per standard Drop-seq protocol ; Macasko et al., 2015). cDNA was created from the captured RNA targets and amplified. Nextera library preparation was used to tagment and index the cDNA for next generation sequencing.

我们开发了一种可调连接技术，用于改造用于捕获单细胞RNA测序（如Drop-seq）所需mRNA的微珠，并将该技术命名为基于单细胞测序的液滴辅助RNA靶向技术（droplet assisted RNA targeting by single cell sequencing，简称DART-seq）。
本研究中，我们将该新技术用于捕获感染小鼠L929细胞的重组呼肠孤病毒3型迪林株（recombinant type 3 Dearing orthoreovirus）的病毒基因组转录本。除捕获靶向病毒基因转录本外，我们还可同时从L929细胞中富集多聚腺苷酸化mRNA（polyadenylated mRNA），以解析宿主-病原体互作（host-pathogen interactions）关系。此外，我们还将该技术用于高效检测一批人外周血单个核细胞（peripheral blood mononuclear cells，PBMCs）样本中B淋巴细胞的天然配对重链和轻链扩增子。

实验整体设计：
商业化的Drop-seq微珠（Chemgene公司）通过可调连接化学方法进行改造，使其在保留捕获多聚腺苷酸化mRNA（polyadenylated mRNA）能力的同时，可靶向捕获目标序列。将改造后的微珠或原始微珠重悬于裂解缓冲液（lysis buffer）中，随后利用微流控设备（microfluidic device）将其与单细胞（感染后的小鼠L929细胞，或另一实验中的人PBMCs）进行共包裹（操作遵循标准Drop-seq实验方案；Macasko等，2015）。从捕获的RNA靶标中合成互补DNA（complementary DNA, cDNA）并进行扩增。采用Nextera文库制备方法对cDNA进行片段化并添加索引标签，以用于下一代测序（next generation sequencing, NGS）。