TY - JOUR T1 - mcSCRB-seq: sensitive and powerful single-cell RNA sequencing JF - bioRxiv DO - 10.1101/188367 SP - 188367 AU - Johannes W. Bagnoli AU - Christoph Ziegenhain AU - Aleksandar Janjic AU - Lucas E. Wange AU - Beate Vieth AU - Swati Parekh AU - Johanna Geuder AU - Ines Hellmann AU - Wolfgang Enard Y1 - 2017/01/01 UR - http://biorxiv.org/content/early/2017/10/18/188367.abstract N2 - Single-cell RNA sequencing (scRNA-seq) has emerged as the central genome-wide method to characterize cellular identities and processes. While performance of scRNA-seq methods is improving, an optimum in terms of sensitivity, cost-efficiency and flexibility has not yet been reached. Among the flexible plate-based methods “Single-Cell RNA-Barcoding and Sequencing” (SCRB-seq) is one of the most sensitive and efficient ones. Based on this protocol, we systematically evaluated experimental conditions such as reverse transcriptases, reaction enhancers and PCR polymerases. We 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 a new scRNA-seq library protocol we call “molecular crowding SCRB-seq” (mcSCRB-seq), which we show to be the most sensitive and one of the most efficient and flexible scRNA-seq methods to date. ER -