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URL: https://dx.doi.org/DOI:10.17504/protocols.io.p9kdr4w

Authors: Johannes Bagnoli, Christoph Ziegenhain, Aleksandar Janjic, Lucas Esteban Wange, Beate Vieth, Swati Parekh, Johanna Geuder, Ines Hellmann, Wolfgang Enard

Group: Human Cell Atlas Method Development Community

Summary: 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 one of the most sensitive and efficient ones. Here, we systematically evaluated 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 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.

Associated Publications: Johannes W. Bagnoli, et al (2018) Sensitive and powerful single-cell RNA sequencing using mcSCRB-seq. Nature Communications 9:2937. doi: 10.1038/s41467-018-05347-6

Affiliations: Ludwig-Maximilians-Universität München, Ludwig-Maximilians-Universität München, Ludwig-Maximilians-Universität München, Ludwig-Maximilians-Universität München, Ludwig-Maximilians-Universität München, Ludwig-Maximilians-Universität München, Ludwig-Maximilians-Universität München, Ludwig-Maximilians-Universität München, Ludwig-Maximilians-Universität München

External URL: https://www.nature.com/articles/s41467-018-05347-6

Version: 2

Publication Date: 2018

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