Xu Ping, Yuan Zhiheng, Lu Xiaohua, Zhou Peng, Qiu Ding, Qiao Zhenghao, Zhou Zhongcheng, Guan Li, Jia Yongkang, He Xuan, Sun Ling, Wan Youzhong, Wang Ming, Yu Yang
China-Japan Union Hospital of Jilin University, Jilin University, Changchun 130033, China.
School of Life Sciences, Jilin University, Changchun 130012, China.
Genomics Proteomics Bioinformatics. 2024 Dec 3;22(5). doi: 10.1093/gpbjnl/qzae072.
Single-cell RNA sequencing (scRNA-seq) has transformed our understanding of cellular diversity with unprecedented resolution. However, many current methods are limited in capturing full-length transcripts and discerning strand orientation. Here, we present RAG-seq, an innovative strand-specific total RNA sequencing technique that combines not-so-random (NSR) primers with Tn5 transposase-mediated tagmentation. RAG-seq overcomes previous limitations by delivering comprehensive transcript coverage and maintaining strand orientation, which are essential for accurate quantification of overlapping genes and detection of antisense transcripts. Through optimized reverse transcription with oligo-dT primers, rRNA depletion via Depletion of Abundant Sequences by Hybridization (DASH), and linear amplification, RAG-seq enhances sensitivity and reproducibility, especially for low-input samples and single cells. Application to mouse oocytes and early embryos highlights RAG-seq's superior performance in identifying stage-specific antisense transcripts, shedding light on their regulatory roles during early development. This advancement represents a significant leap in transcriptome analysis within complex biological contexts.
单细胞RNA测序（scRNA-seq）以前所未有的分辨率改变了我们对细胞多样性的理解。然而，许多当前的方法在捕获全长转录本和辨别链方向方面存在局限性。在此，我们介绍RAG-seq，一种创新的链特异性全RNA测序技术，它将非随机（NSR）引物与Tn5转座酶介导的转座标签法相结合。RAG-seq通过提供全面的转录本覆盖并保持链方向克服了先前的局限性，这对于重叠基因的准确量化和反义转录本的检测至关重要。通过使用寡聚dT引物进行优化的逆转录、通过杂交去除丰富序列（DASH）进行rRNA去除以及线性扩增，RAG-seq提高了灵敏度和可重复性，特别是对于低输入样本和单细胞。在小鼠卵母细胞和早期胚胎中的应用突出了RAG-seq在识别阶段特异性反义转录本方面的卓越性能，揭示了它们在早期发育过程中的调控作用。这一进展代表了复杂生物学背景下转录组分析的重大飞跃。
