@article{203c985160ef4bb9a415baf4bdbf748e,
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.}",
note = "Funding Information: R.S.L. was funded by NIH-1RC1 HG005471, NIH-2R01 HG003647; R.S.L. and F.H.G. by Transformative R01MH095741; F.H.G. by the Jeffry M. and Barbara Picower Foundation, the Mathers Foundation, the McDonnell Foundation and the National Institute of Mental Health (NIMH); and M.J.M. by a Crick-Jacobs Junior Fellowship. K.B. is supported by the National Science Foundation Graduate Research Fellowship under grant no. DGE-1144086. The authors thank the Allen Institute for Brain Science founders, Paul G. Allen and Jody Allen, for their vision, encouragement and support. Research was supported in part by the Allen Institute for Brain Science. Publisher Copyright: {\textcopyright} 2016 Nature America, Inc.",
year = "2016",
month = mar,
day = "1",
doi = "10.1038/nprot.2016.015",
language = "English (US)",
volume = "11",
pages = "499--524",
journal = "Nature Protocols",
issn = "1754-2189",
publisher = "Nature Publishing Group",
number = "3",
}