Journal Article

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

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

2016

Overview

This protocol describes how to sequence the transcriptome from a single nucleus. It is particularly suited to cell types that are difficult to isolate as intact whole cells, such as neurons. 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.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 631/1647/2217/2018 / 631/1647/514/1949 / 631/208/212/2019 / 631/378/340 / Analytical Chemistry / Autopsy - methods / Biological Techniques / Brain - cytology / Brain damage / Cell Fractionation - methods / Cell nuclei / Cell Nucleolus - genetics / Cells / Computational Biology/Bioinformatics / Data analysis / DNA, Complementary - genetics / Flow cytometry / Gene expression / Gene Expression Profiling - methods / Gene Library / Genetic aspects / Homogenization / Humans / Life Sciences / Methods / Microarrays / Neurons / Neurons - metabolism / Nuclei (cytology) / Organic Chemistry / Protocol / Ribonucleic acid / RNA / RNA - genetics / RNA sequencing / Sequence Analysis, RNA - methods / Single-Cell Analysis - methods / Tissue Preservation / Transcriptome / Transcriptomes