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.
Researchers at Southeast University, Nanjing have developed a highly sensitive library construction protocol for ultralow input RNA sequencing (ulRNA-seq). The researchers 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.
Validation of ulRNA-seq protocol using single-cell micro-region
A The number of genes detected Smart-seq2 and ulRNA-seq protocol. B The number of genes detected in different expression levels binned by standardized expression FPKM in single-cell. C The ratio of the detected genes in the cell marker gene database of mice. D Sensitivity for detecting genes in single-cell. E Precision for detecting genes in single-cell. F Heatmap showing person correlation between the five replicates
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. The researchers expect that ulRNA-seq will sequence and transcriptome characterization for the subcellular of disease tissue, to find the corresponding treatment plan.