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Nat Protoc. 2016 Mar;11(3):499-524. doi: 10.1038/nprot.2016.015. Epub 2016 Feb 18.

Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons.

Author information

1
J. Craig Venter Institute, La Jolla, California, USA.
2
Institute of Microbiology, ETH Zurich, Zurich, Switzerland.
3
J. Craig Venter Institute, Rockville, Maryland, USA.
4
Salk Institute for Biological Studies, La Jolla, California, USA.
5
Allen Institute for Brain Science, Seattle, Washington, USA.
6
Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain.
7
LeGene Biosciences, San Diego, California, USA.
8
Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia, USA.

Abstract

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.

PMID:
26890679
PMCID:
PMC4941947
DOI:
10.1038/nprot.2016.015
[Indexed for MEDLINE]
Free PMC Article

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