计算生物学
核糖核酸
RNA序列
cDNA文库
生物
灵活性(工程)
互补DNA
基因组文库
计算机科学
转录组
基因
遗传学
基序列
基因表达
统计
数学
作者
Johannes Bagnoli,Christoph Ziegenhain,Aleksandar Janjic,Lucas E. Wange,Beate Vieth,Swati Parekh,Johanna Geuder,Ines Hellmann,Wolfgang Enard
标识
DOI:10.1038/s41467-018-05347-6
摘要
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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