Jia Erteng, Shi Huajuan, Wang Ying, Zhou Ying, Liu Zhiyu, Pan Min, Bai Yunfei, Zhao Xiangwei, Ge Qinyu
State Key Laboratory of Bioelectronics, School of Biological Science & Medical Engineering, Southeast University, Nanjing, 210096, China.
School of Medicine, Southeast University, Nanjing, 210097, China.
BMC Genomics. 2021 Nov 10;22(1):809. doi: 10.1186/s12864-021-08132-w.
Single-cell RNA sequencing (scRNA-seq) provides new insights to address biological and medical questions, and it will benefit more from the ultralow input RNA or subcellular sequencing.
Here, we present a highly sensitive library construction protocol for ultralow input RNA sequencing (ulRNA-seq). We systematically evaluate experimental conditions of this protocol, such as reverse transcriptase, template-switching oligos (TSO), and template RNA structure. It was found that Maxima H Minus reverse transcriptase and rN modified TSO, as well as all RNA templates capped with m7G improved the sequencing sensitivity and low abundance gene detection ability. RNA-seq libraries were successfully prepared from total RNA samples as low as 0.5 pg, and more than 2000 genes have been identified.
The ability of low abundance gene detection and sensitivity were largely enhanced with this optimized protocol. It was also confirmed in single-cell sequencing, that more genes and cell markers were identified compared to conventional sequencing method. We expect that ulRNA-seq will sequence and transcriptome characterization for the subcellular of disease tissue, to find the corresponding treatment plan.
单细胞RNA测序（scRNA-seq）为解决生物学和医学问题提供了新的见解，并且它将从超低输入RNA或亚细胞测序中受益更多。
在此，我们提出了一种用于超低输入RNA测序（ulRNA-seq）的高灵敏度文库构建方案。我们系统地评估了该方案的实验条件，如逆转录酶、模板转换寡核苷酸（TSO）和模板RNA结构。发现Maxima H Minus逆转录酶和rN修饰的TSO，以及所有用m7G加帽的RNA模板提高了测序灵敏度和低丰度基因检测能力。从低至0.5 pg的总RNA样本中成功制备了RNA-seq文库，并鉴定出了2000多个基因。
通过这种优化方案，低丰度基因检测能力和灵敏度得到了很大提高。在单细胞测序中也得到证实，与传统测序方法相比，鉴定出了更多的基因和细胞标志物。我们期望ulRNA-seq将对疾病组织的亚细胞进行测序和转录组表征，以找到相应的治疗方案。
