Optimization of library preparation based on SMART for ultralow RNA-seq in mice brain tissues

BMC Genomics

Min Pan¹  Huajuan Shi²  Xiangwei Zhao²  Qinyu Ge²  Zhiyu Liu²  Erteng Jia²  Ying Zhou²  Ying Wang²  Yunfei Bai² 

[1] School of Medicine, Southeast University, 210097, Nanjing, China;State Key Laboratory of Bioelectronics, School of Biological Science & Medical Engineering, Southeast University, 210096, Nanjing, China;

关键词: scRNA-seq;    Sensitivity;    Template-switching oligos terminal modification;    Low abundance gene detection;    Subcellular;   

DOI  :  10.1186/s12864-021-08132-w

来源: Springer

【 摘 要 】

BackgroundSingle-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.ResultsHere, 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.ConclusionsThe 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.

【 授权许可】

CC BY