Improvements to Single-cell mRNA Sequencing Technology

Single-cell RNA sequencing (scRNA-seq) is an important tool for researchers to understand the variability in gene expression at the cell-to-cell level. scRNA-seq is a widely used protocol, but a major drawback of this laboratory technique is its high costs. My project involved lowering the costs of an RNA sequencing protocol, CEL-Seq2, while maintaining a high level of sensitivity, the ability to uniquely identify a sequence. CEL-Seq2 sequencing involves first reading a cell-specific barcode, then the unique gene sequence. Our new protocol, CEL-Seq3, involves 1) a reversal of this procedure, such that the unique gene sequence is read prior to the cell barcode, and 2) lowering the sequence read length. Preliminary results on the simulation showed that the cost of sequencing was reduced from $3200 to $1700 per run. I first conducted a computer-based simulation to test if a reduction of sequencing length would affect sensitivity. A Pearson correlation of greater than 0.8 was obtained when previously attained scRNA-seq data was trimmed. This high correlation value shows that sensitivity is not reduced with our proposed changes. Following these results, two successful sequencing runs have been conducted with this reduction. This data shows that the CEL-Seq3 protocol has the potential to provide a cost-effective method for identifying gene expression in single cells, allowing scientists to study embryonic development and understand diseases, to name a few applications.