LAST-seq:single-cell RNA sequencing by direct amplification of single-stranded RNA without prior reverse transcription and second-strand synthesis

Existing single-cell RNA sequencing (scRNA-seq) methods rely on reverse transcription (RT) and second-strand synthesis (SSS) to convert single-stranded RNA into double-stranded DNA prior to amplification, with the limited RT/SSS efficiency compromising RNA detectability. Here, we develop a new scRNA-seq method, Linearly Amplified Single-stranded-RNA-derived Transcriptome sequencing (LAST-seq), which directly amplifies the original single-stranded RNA molecules without prior RT/SSS. LAST-seq offers a high single-molecule capture efficiency and a low level of technical noise for single-cell transcriptome analyses. Using LAST-seq, we characterize transcriptional bursting kinetics in human cells, revealing a role of topologically associating domains in transcription regulation. HEK293T and eHAP1 cells were sorted by Fluorescence-activated cell sorting (FACS) and analyzed using scRNAseq.

现有单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）方法通常依赖逆转录（reverse transcription, RT）与第二链合成（second-strand synthesis, SSS），在扩增前将单链RNA转化为双链DNA；但逆转录与第二链合成的效率有限，会降低RNA的可检测性。本研究开发了一种新型scRNA-seq方法——线性扩增单链RNA衍生转录组测序（Linearly Amplified Single-stranded-RNA-derived Transcriptome sequencing, LAST-seq），该方法无需预先进行逆转录与第二链合成，可直接对原始单链RNA分子进行扩增。LAST-seq可实现较高的单分子捕获效率，并在单细胞转录组分析中保持较低的技术噪声水平。借助LAST-seq，本研究对人类细胞的转录爆发动力学特征进行了表征，揭示了拓扑关联结构域在转录调控中的作用。研究人员通过荧光激活细胞分选（Fluorescence-activated cell sorting, FACS）对HEK293T与eHAP1细胞进行分选，并利用scRNA-seq完成分析。