Hayashi Tetsutaro, Ozaki Haruka, Sasagawa Yohei, Umeda Mana, Danno Hiroki, Nikaido Itoshi
Bioinformatics Research Unit, Advanced Center for Computing and Communication, RIKEN, 2-1 Hirosawa Wako, Saitama, 351-0198, Japan.
Single-cell Omics Research Unit, Center for RIKEN Center for Developmental Biology, RIKEN, 2-1 Hirosawa Wako, Saitama, 351-0198, Japan.
Nat Commun. 2018 Feb 12;9(1):619. doi: 10.1038/s41467-018-02866-0.
Total RNA sequencing has been used to reveal poly(A) and non-poly(A) RNA expression, RNA processing and enhancer activity. To date, no method for full-length total RNA sequencing of single cells has been developed despite the potential of this technology for single-cell biology. Here we describe random displacement amplification sequencing (RamDA-seq), the first full-length total RNA-sequencing method for single cells. Compared with other methods, RamDA-seq shows high sensitivity to non-poly(A) RNA and near-complete full-length transcript coverage. Using RamDA-seq with differentiation time course samples of mouse embryonic stem cells, we reveal hundreds of dynamically regulated non-poly(A) transcripts, including histone transcripts and long noncoding RNA Neat1. Moreover, RamDA-seq profiles recursive splicing in >300-kb introns. RamDA-seq also detects enhancer RNAs and their cell type-specific activity in single cells. Taken together, we demonstrate that RamDA-seq could help investigate the dynamics of gene expression, RNA-processing events and transcriptional regulation in single cells.
全转录组RNA测序已被用于揭示聚腺苷酸（poly(A)）和非聚腺苷酸（non-poly(A)）RNA的表达、RNA加工及增强子活性。尽管单细胞全转录组RNA测序技术在单细胞生物学领域具有潜在应用价值，但迄今为止尚未开发出相关方法。在此，我们描述了随机位移扩增测序（RamDA-seq），这是首个用于单细胞的全转录组RNA测序方法。与其他方法相比，RamDA-seq对非聚腺苷酸RNA具有高灵敏度，且转录本全长覆盖率近乎完整。利用RamDA-seq对小鼠胚胎干细胞分化时间进程样本进行分析，我们发现了数百个动态调控的非聚腺苷酸转录本，包括组蛋白转录本和长链非编码RNA Neat1。此外，RamDA-seq还能分析超过300 kb内含子中的递归剪接。RamDA-seq还可在单细胞中检测增强子RNA及其细胞类型特异性活性。综上所述，我们证明RamDA-seq有助于研究单细胞中基因表达、RNA加工事件及转录调控的动态变化。
