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.

The BRAIN Initiative Cell Census Consortium: Lessons Learned toward Generating a Comprehensive Brain Cell Atlas

“…Single-nucleus RNA-sequencing (variously referred to as snRNA-seq, sNuc-seq, Div-Seq, DroNc-seq) has its challenges in that nuclear RNA is less abundant than cellular RNA, and includes precursor RNAs containing introns in various stages of processing. However, results similar to whole-cell RNA-seq have recently been reported, indicating that single-nucleus RNA-seq is a viable alternative for many experimental designs (Habib et al, 2017; Habib et al, 2016; Krishnaswami et al, 2016; Lacar et al, 2016; Lake et al, 2016). Work will need to be done to harmonize human data with data from mouse and other species where fresh tissue and whole cell RNA-seq will be performed.…”

Cerebral cortical neuron diversity and development at single-cell resolution

“…There are, however, reliable and reproducible protocols for isolating single neuronal nuclei from frozen postmortem human brain[36,37]. Fortunately, the nucleus contains a significant amount of messenger RNA, and several studies have now demonstrated single-nucleus RNA sequencing[38–40]. Lake et al have taken this approach for scRNA-seq–based cell type classification in the human cerebral cortex, identifying 16 neuronal subtypes – 8 excitatory and 8 inhibitory[34].…”

Epigenetic Research in Neuropsychiatric Disorders: the “Tissue Issue”

“…Also, for some disorders such as autism and schizophrenia, connectivity between brain regions may be of primary importance, rather than specific regional states [18]. Therefore, an argument for tissue specificity should really be cast as an argument for brain region—and cell type—specificity, and current approaches to brain epigenetic measurement are only beginning to reach this granularity [19, 20]. With respect to cell type, a portion of epigenomic marks are known to be cell-type specific, and the brain is a heterogeneous mix of neurons and glia, including astrocytes, oligodendrocytes, and microglia.…”