single-cell RNA-seq of murine spleen by TAS-Seq

Single-cell RNA-sequencing (scRNA-seq) is valuable for analyzing cellular heterogeneity. Cell composition accuracy is critical for analyzing cell-cell interaction networks from scRNA-seq data because cell abundancy might affect to the network. We developed terminator-assisted solid-phase cDNA amplification and sequencing (TAS-Seq), a scRNA-seq method that relied on terminator, terminal transferase, and nanowell/beads-based scRNA-seq platform, that could acquire scRNA-seq data with higher correlation with flow-cytometric data, gene-detection sensitivity, and robustness than widely-used methods. To clarify performance of TAS-Seq, we analyzed mouse spleen cells collected from subcutaneous lewis lung carcinoma (LLC) model. 5x105 LLC cells were subcutaneously injected to 10 week old C57BL/6J female mice, treated with 200 μg/shot anti-CD4 antibody at on day 5 and day 9 post injection, and spleen were recovered at day 12. Resulted spleen cells were subjected by BD Rhapsody workflow, and half of BD Rhapsody beads were processed by TAS-Seq. and the other half of the beads were processed by BD official WTA protocol.

单细胞RNA测序（single-cell RNA-sequencing, scRNA-seq）在解析细胞异质性领域具有重要应用价值。从scRNA-seq数据中分析细胞互作网络时，细胞组成准确性至关重要，因为细胞丰度可能对该网络产生影响。本研究开发了终止子辅助固相cDNA扩增测序（terminator-assisted solid-phase cDNA amplification and sequencing, TAS-Seq）——一种依托终止子、末端脱氧核苷酸转移酶以及基于纳米孔/磁珠的scRNA-seq平台的单细胞RNA测序方法——相较于主流测序方法，该方法可获得与流式细胞术数据相关性更高、基因检测灵敏度更优且鲁棒性更强的scRNA-seq数据。为明确TAS-Seq的性能表现，本研究对取自皮下路易斯肺癌（Lewis lung carcinoma, LLC）模型的小鼠脾脏细胞开展了分析实验：向10周龄雌性C57BL/6J小鼠皮下注射5×10⁵个LLC细胞，于注射后第5天和第9天以200 μg/次的剂量注射抗CD4抗体，并于注射后第12天摘取小鼠脾脏。将所得脾脏细胞按照BD Rhapsody测序流程进行处理，将其中一半磁珠采用TAS-Seq进行测序分析，另一半磁珠则采用BD官方WTA方案进行测序分析。