Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons.
Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons.
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DOI：
10.1038/nprot.2016.015
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发表时间：
2016-03
期刊：
影响因子：
14.8
通讯作者：
Lasken RS
中科院分区：
文献类型：
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作者：
Krishnaswami SR;Grindberg RV;Novotny M;Venepally P;Lacar B;Bhutani K;Linker SB;Pham S;Erwin JA;Miller JA;Hodge R;McCarthy JK;Kelder M;McCorrison J;Aevermann BD;Fuertes FD;Scheuermann RH;Lee J;Lein ES;Schork N;McConnell MJ;Gage FH;Lasken RS
描述了用于对细胞核的转录组进行测序的方案。从样本中分离细胞核并通过FACS分选，构建cDNA文库并进行RNA-seq，然后进行数据分析。一些步骤遵循公开的方法（用于cDNA合成的Smart-seq 2和Nextera XT条形码化文库制备），在此不详细描述。以前用于组织RNA-seq的单细胞方法包括在30 °C下使用蛋白酶处理的细胞解离，已知其改变转录组。我们在4 °C下从组织匀浆中分离细胞核，这会造成最小的损伤。核转录组可以从储存在−80 °C的死后人脑组织中获得，使大脑档案可以用于单个神经元的RNA测序。该方法还允许研究细胞核特有的生物学特征，例如某些转录物和某些非编码RNA的前体的富集。按照这个程序，大约需要4天来构建准备测序的cDNA文库。
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.