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J Vis Exp. 2018 Oct 12;(140). doi: 10.3791/58413.

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing.

Author information

1
Spinal Circuits and Plasticity Unit, National Institute of Neurological Disorders and Stroke.
2
Bioinformatics Section, Information Technology Program, National Institute of Neurological Disorders and Stroke.
3
Laboratory of Cochlear Development, National Institute on Deafness and Other Communication Disorders; Single Cell Analysis Facility, Frederick National Laboratory.
4
Laboratory of Cochlear Development, National Institute on Deafness and Other Communication Disorders.
5
Spinal Circuits and Plasticity Unit, National Institute of Neurological Disorders and Stroke; ariel.levine@nih.gov.

Abstract

Probing an individual cell's gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

PMID:
30371670
PMCID:
PMC6235529
[Available on 2019-10-12]
DOI:
10.3791/58413
[Indexed for MEDLINE]
Free PMC Article

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