Sensitive and powerful single-cell RNA sequencing using mcSCRB-seq
Single-cell RNA sequencing (scRNA-seq) has emerged as a central genome-wide method to characterize cellular identities and processes. Consequently, improving its sensitivity, flexibility and cost-efficiency can advance many research questions. Among the flexible plate-based methods, “Single-Cell RNA-Barcoding and Sequencing” (SCRB-seq) is highly sensitive and efficient. Here, we systematically evaluate experimental conditions of this protocol and find that adding polyethylene glycol considerably increases sensitivity by enhancing cDNA synthesis. Furthermore, using Terra polymerase increases efficiency due to a more even cDNA amplification that requires less sequencing of libraries. We combined these and other improvements to develop a new scRNA-seq library protocol we call “molecular crowding SCRB-seq” (mcSCRB-seq), which we show to be one of the most sensitive, efficient and flexible scRNA-seq methods to date.