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 -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.
authors.affiliation: J. Craig Venter Institute, La Jolla, California, USA.
authors.forename: Suguna Rani
authors.initials: SR
authors.lastname: Krishnaswami
authors.affiliation: Institute of Microbiology, ETH Zurich, Zurich, Switzerland.
authors.forename: Rashel V
authors.initials: RV
authors.lastname: Grindberg
authors.affiliation: J. Craig Venter Institute, La Jolla, California, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Novotny
authors.affiliation: J. Craig Venter Institute, Rockville, Maryland, USA.
authors.forename: Pratap
authors.initials: P
authors.lastname: Venepally
authors.affiliation: Salk Institute for Biological Studies, La Jolla, California, USA.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Lacar
authors.affiliation: J. Craig Venter Institute, La Jolla, California, USA.
authors.forename: Kunal
authors.initials: K
authors.lastname: Bhutani
authors.affiliation: Salk Institute for Biological Studies, La Jolla, California, USA.
authors.forename: Sara B
authors.initials: SB
authors.lastname: Linker
authors.affiliation: Salk Institute for Biological Studies, La Jolla, California, USA.
authors.forename: Son
authors.initials: S
authors.lastname: Pham
authors.affiliation: Salk Institute for Biological Studies, La Jolla, California, USA.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Erwin
authors.affiliation: Allen Institute for Brain Science, Seattle, Washington, USA.
authors.forename: Jeremy A
authors.initials: JA
authors.lastname: Miller
authors.affiliation: Allen Institute for Brain Science, Seattle, Washington, USA.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Hodge
authors.affiliation: J. Craig Venter Institute, La Jolla, California, USA.
authors.forename: James K
authors.initials: JK
authors.lastname: McCarthy
authors.affiliation: Salk Institute for Biological Studies, La Jolla, California, USA.
authors.forename: Martin
authors.initials: M
authors.lastname: Kelder
authors.affiliation: J. Craig Venter Institute, La Jolla, California, USA.
authors.forename: Jamison
authors.initials: J
authors.lastname: McCorrison
authors.affiliation: J. Craig Venter Institute, La Jolla, California, USA.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Aevermann
authors.affiliation: J. Craig Venter Institute, La Jolla, California, USA.|Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Francisco Diez
authors.initials: FD
authors.lastname: Fuertes
authors.affiliation: J. Craig Venter Institute, La Jolla, California, USA.
authors.forename: Richard H
authors.initials: RH
authors.lastname: Scheuermann
authors.affiliation: LeGene Biosciences, San Diego, California, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Lee
authors.affiliation: Allen Institute for Brain Science, Seattle, Washington, USA.
authors.forename: Ed S
authors.initials: ES
authors.lastname: Lein
authors.affiliation: J. Craig Venter Institute, La Jolla, California, USA.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Schork
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: McConnell
authors.affiliation: Salk Institute for Biological Studies, La Jolla, California, USA.
authors.forename: Fred H
authors.initials: FH
authors.lastname: Gage
authors.affiliation: J. Craig Venter Institute, La Jolla, California, USA.
authors.forename: Roger S
authors.initials: RS
authors.lastname: Lasken
mesh_terms: D001344:Autopsy / Q000379:methods; D001921:Brain / Q000166:cytology*; D002458:Cell Fractionation / Q000379:methods; D002466:Cell Nucleolus / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods; D015723:Gene Library; D006801:Humans; D009474:Neurons / Q000378:metabolism*; D012313:RNA / Q000235:genetics*; D017423:Sequence Analysis, RNA / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods; D014021:Tissue Preservation; D059467:Transcriptome
chemical_list: D018076:DNA, Complementary; D012313:RNA