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Sensitive and powerful single-cell RNA sequencing using mcSCRB-seq.

ABSTRACT: 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 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.

SUBMITTER: Bagnoli JW

PROVIDER: S-EPMC6062574 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Sensitive and powerful single-cell RNA sequencing using mcSCRB-seq.

Bagnoli Johannes W JW Ziegenhain Christoph C Janjic Aleksandar A Wange Lucas E LE Vieth Beate B Parekh Swati S Geuder Johanna J Hellmann Ines I Enard Wolfgang W

Nature communications 20180726 1

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 ...[more]

PMID: 30050112

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Sensitive and powerful single-cell RNA sequencing using mcSCRB-seq

Project description: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 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.

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