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

Suguna Rani Krishnaswami, Rashel V. Grindberg, Mark Novotny, Pratap Venepally, Benjamin Lacar, Kunal Bhutani, Sara B. Linker, Son Pham, Jennifer A. Erwin, Jeremy A. Miller, Rebecca Hodge, James K. McCarthy, Martin Kelder, Jamison McCorrison, Brian D. Aevermann, Francisco Diez Fuertes, Richard H. Scheuermann, Jun Lee, Ed S. Lein, Nicholas SchorkMichael J. McConnell, Fred H. Gage, Roger S. Lasken

Research output: Contribution to journalArticle

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 â '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.

Original languageEnglish (US)
Pages (from-to)499-524
Number of pages26
JournalNature protocols
Volume11
Issue number3
DOIs
StatePublished - Mar 1 2016
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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  • Cite this

    Krishnaswami, S. R., Grindberg, R. V., Novotny, M., Venepally, P., Lacar, B., Bhutani, K., Linker, S. B., Pham, S., Erwin, J. A., Miller, J. A., Hodge, R., McCarthy, J. K., Kelder, M., McCorrison, J., Aevermann, B. D., Fuertes, F. D., Scheuermann, R. H., Lee, J., Lein, E. S., ... Lasken, R. S. (2016). Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons. Nature protocols, 11(3), 499-524. https://doi.org/10.1038/nprot.2016.015