RNA-sequencing from single nuclei

It has recently been established that synthesis of double-stranded cDNA can be done from a single cell for use in DNA sequencing. Global gene expression can be quantified from the number of reads mapping to each gene, and mutations and mRNA splicing variants determined from the sequence reads. Here...

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Published in:	Proceedings of the National Academy of Sciences - PNAS Vol. 110; no. 49; pp. 19802 - 19807
Main Authors:	Grindberg, Rashel V., Yee-Greenbaum, Joyclyn L., McConnell, Michael J., Novotny, Mark, O'Shaughnessy, Andy L., Lambert, Georgina M., Araúzo-Bravo, Marcos J., Lee, Jun, Fishman, Max, Robbins, Gillian E., Lin, Xiaoying, Venepally, Pratap, Badger, Jonathan H., Galbraith, David W., Gage, Fred H., Lasken, Roger S.
Format:	Journal Article
Language:	English
Published:	United States National Academy of Sciences 03-12-2013 NATIONAL ACADEMY OF SCIENCES National Acad Sciences
Subjects:	Animals Biological Sciences Brain Cell lines Cell nucleus Cell Nucleus - genetics Cell Nucleus - metabolism Cells Complementary DNA Dentate Gyrus - cytology Embryonic Stem Cells Flow Cytometry Gene expression Gene Expression Profiling - methods Genes High-Throughput Nucleotide Sequencing - methods Mice Micromanipulation Microscopy, Fluorescence Neural Stem Cells - metabolism Neurons Pluripotent stem cells Ribonucleic acid RNA Rodents Stem cells Tissues deep sequencing nuclear RNA whole cell RNA
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Summary:	It has recently been established that synthesis of double-stranded cDNA can be done from a single cell for use in DNA sequencing. Global gene expression can be quantified from the number of reads mapping to each gene, and mutations and mRNA splicing variants determined from the sequence reads. Here we demonstrate that this method of transcriptomic analysis can be done using the extremely low levels of mRNA in a single nucleus, isolated from a mouse neural progenitor cell line and from dissected hippocampal tissue. This method is characterized by excellent coverage and technical reproducibility. On average, more than 16,000 of the 24,057 mouse protein-coding genes were detected from single nuclei, and the amount of gene-expression variation was similar when measured between single nuclei and single cells. Several major advantages of the method exist: first, nuclei, compared with whole cells, have the advantage of being easily isolated from complex tissues and organs, such as those in the CNS. Second, the method can be widely applied to eukaryotic species, including those of different kingdoms. The method also provides insight into regulatory mechanisms specific to the nucleus. Finally, the method enables dissection of regulatory events at the single-cell level; pooling of 10 nuclei or 10 cells obscures some of the variability measured in transcript levels, implying that single nuclei and cells will be extremely useful in revealing the physiological state and interconnectedness of gene regulation in a manner that avoids the masking inherent to conventional transcriptomics using bulk cells or tissues.
Bibliography:	http://dx.doi.org/10.1073/pnas.1319700110 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Contributed by Fred H. Gage, October 23, 2013 (sent for review August 12, 2013) Author contributions: D.W.G. and R.S.L. conceived the project; D.W.G., F.H.G., and R.S.L. designed research; R.V.G., J.L.Y.-G., and R.S.L. developed experimental designs; M.J.M. and M.N. developed flow cytometry methods; R.V.G., J.L.Y.-G., M.J.M., G.M.L., M.F., and G.E.R. performed research; R.V.G., J.L.Y.-G., M.J.M., G.M.L., M.F., and G.E.R. performed sample isolation, cDNA synthesis, and quantitative PCR assays; M.J.M. and F.H.G developed the NPC cell line and prepared nuclei from NPCs and mouse brain; M.J.A.-B., P.V., and J.H.B. performed bioinformatic analyses; M.N. contributed new reagents/analytic tools; A.L.O. contributed to library production and sequencing; J.L.Y.-G., D.W.G., and F.H.G. contributed to data analysis; X.L. provided bioinformatic expertise in SOLiD whole-transcriptome data analysis; R.V.G., J.L.Y.-G., M.J.M., A.L.O., M.J.A.-B., J.L., X.L., P.V., J.H.B., D.W.G., F.H.G., and R.S.L. analyzed data; and R.V.G., D.W.G., and R.S.L. wrote the paper. 2J.L.Y.-G. and M.J.M. contributed equally to this paper. 1Present address: Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, 48149 Münster, Germany. 3Present address: Applied Biosystems, Life Technologies, Foster City, CA 94404.
ISSN:	0027-8424 1091-6490
DOI:	10.1073/pnas.1319700110