Krishnaswami Suguna Rani, Grindberg Rashel V, Novotny Mark, Venepally Pratap, Lacar Benjamin, Bhutani Kunal, Linker Sara B, Pham Son, Erwin Jennifer A, Miller Jeremy A, Hodge Rebecca, McCarthy James K, Kelder Martin, McCorrison Jamison, Aevermann Brian D, Fuertes Francisco Diez, Scheuermann Richard H, Lee Jun, Lein Ed S, Schork Nicholas, McConnell Michael J, Gage Fred H, Lasken Roger S
J. Craig Venter Institute, La Jolla, California, USA.
Institute of Microbiology, ETH Zurich, Zurich, Switzerland.
Nat Protoc. 2016 Mar;11(3):499-524. doi: 10.1038/nprot.2016.015. Epub 2016 Feb 18.
A protocol is described for sequencing the transcriptome of a cell nucleus. Nuclei are isolated from specimens and sorted by FACS, cDNA libraries are constructed and RNA-seq is performed, followed by data analysis. Some steps follow published methods (Smart-seq2 for cDNA synthesis and Nextera XT barcoded library preparation) and are not described in detail here. Previous single-cell approaches for RNA-seq from tissues include cell dissociation using protease treatment at 30 °C, which is known to alter the transcriptome. We isolate nuclei at 4 °C from tissue homogenates, which cause minimal damage. Nuclear transcriptomes can be obtained from postmortem human brain tissue stored at -80 °C, making brain archives accessible for RNA-seq from individual neurons. The method also allows investigation of biological features unique to nuclei, such as enrichment of certain transcripts and precursors of some noncoding RNAs. By following this procedure, it takes about 4 d to construct cDNA libraries that are ready for sequencing.
本文描述了一种用于对细胞核转录组进行测序的方案。从标本中分离细胞核并通过荧光激活细胞分选术（FACS）进行分选，构建cDNA文库并进行RNA测序，随后进行数据分析。有些步骤遵循已发表的方法（用于cDNA合成的Smart-seq2和Nextera XT条形码文库制备），在此不再详细描述。以前从组织中进行RNA测序的单细胞方法包括在30°C下使用蛋白酶处理进行细胞解离，已知这会改变转录组。我们在4°C下从组织匀浆中分离细胞核，这样造成的损伤最小。可以从储存在-80°C的死后人类脑组织中获得核转录组，从而使大脑档案可用于单个神经元的RNA测序。该方法还允许研究细胞核特有的生物学特征，例如某些转录本和一些非编码RNA前体的富集。按照此程序，构建准备好测序的cDNA文库大约需要4天。
