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 Schork; Michael J. McConnell; Fred H. Gage; Roger S. Lasken

doi:10.1038/nprot.2016.015

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

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321 Citations (Scopus)

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 language	English
Pages (from-to)	499-524
Number of pages	26
Journal	Nature Protocols
Volume	11
Issue number	3
DOIs	https://doi.org/10.1038/nprot.2016.015
Publication status	Published - 1 Mar 2016
Externally published	Yes

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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

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title = "Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons",

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 {\^a} '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.",

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

year = "2016",

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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 2016, 'Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons', Nature Protocols, vol. 11, no. 3, pp. 499-524. https://doi.org/10.1038/nprot.2016.015

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AU - Novotny, Mark

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AU - Lacar, Benjamin

AU - Bhutani, Kunal

AU - Linker, Sara B.

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AU - Erwin, Jennifer A.

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AU - Hodge, Rebecca

AU - McCarthy, James K.

AU - Kelder, Martin

AU - McCorrison, Jamison

AU - Aevermann, Brian D.

AU - Fuertes, Francisco Diez

AU - Scheuermann, Richard H.

AU - Lee, Jun

AU - Lein, Ed S.

AU - Schork, Nicholas

AU - McConnell, Michael J.

AU - Gage, Fred H.

AU - Lasken, Roger S.

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

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