Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.
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.
The nervous system is comprised of heterogenous groups of cells that display a diverse array of morphological, biochemical, and electrophysiological properties. While the bulk RNA sequencing has been useful for determining tissue-wide changes in the gene expression under different conditions, it precludes the detection of transcriptional changes at the single-cell level. Recent advances in the single-cell transcriptional analysis have enabled the classification of heterogenous cells into functional groups based on their molecular repertoire and can even be leveraged to detect sets of neurons that had been recently active.1,2,3,4 Over the last ten years, the development of single cell RNA sequencing (scRNA-Seq) has enabled the study of gene expression in individual cells, providing a view into cell-type diversity.5
The emergence of scalable approaches such as massively parallel scRNA-Seq, has provided platforms to sequence heterogeneous tissues, including many regions of the central nervous system.6,7,8,9,10,11,12,13,14,15 However, single cell dissociation methods can lead to the cell death as well as experimental changes in gene expression.16 Recent work has adapted single cell sequencing methods to enable preservation of endogenous transcriptional profiles.1,3,4,17,18,19 These strategies have been particularly suitable for detecting immediate early gene (IEG) expression following sensory stimulus or behavior.3,4 In the future, this strategy could also be used to study dynamic changes in tissues in disease states or in response to stress. Of these methods, single nucleus RNA sequencing (snRNA-Seq) is a promising approach that does not involve stress-inducing cell dissociation and can be used on difficult to dissociate tissue (such as the spinal cord), as well as frozen tissue.4,17,18,19 Adapted from previous nuclei isolation methods,20,21,22,23,25 snRNA-Seq typically utilizes rapid tissue disruption and cell lysis under cold conditions, centrifugation, and separation of nuclei from cellular debris.4 Nuclei can be isolated for the downstream next-generation sequencing on multiple microfluidic droplet encapsulation platforms.4,7,24,25 This method allows for a snapshot of the transcriptional activity of thousands of cells at a moment in time.
There are multiple strategies for releasing nuclei from cells before isolation and sequencing, each with their own advantages and disadvantages. Here, we describe and compare two protocols to enable isolation of nuclei from the adult spinal cord for the downstream massively parallel snRNA-Seq: detergent-mechanical lysis and hypotonic-mechanical lysis. Detergent-mechanical lysis provides complete tissue disruption and a higher final yield of nuclei. Hypotonic mechanical-lysis includes a controllable degree of tissue disruption, providing an opportunity for selecting a balance between the quantity and purity of the final nuclear yield. These approaches provide comparable RNA yield, detected numbers of genes per nucleus, and cell-type profiling and also can both be used successfully for snRNA-Seq.
The ultimate goal of this protocol is to isolate nuclei containing high-quality RNA for downstream transcriptional analysis. We adapted snRNA-Seq methods in order to profile all of the cell types in the spinal cord. Initially, we found that typical cell dissociation methods were ineffective for single cell RNA sequencing, as spinal cord neurons are particularly vulnerable to cell death. Furthermore, cell dissociation methods induce expression of various activity- and stress-response genes by up to several hundred-fold.<s…
The authors have nothing to disclose.
This work was supported by the intramural program of NINDS (1 ZIA NS003153 02) and NIDCD (1 ZIA DC000059 18). We thank L. Li and C.I. Dobrott for their technical support and helpful discussions, and C. Kathe for reviewing the manuscript.
Sucrose | Invitrogen | 15503-022 | |
1 M HEPES (pH = 8.0) | Gibco | 15630-080 | |
CaCl2 | Sigma Aldrich | C1016-100G | |
MgAc | Sigma Aldrich | M1028-10X1ML | |
0.5 M EDTA (pH = 8.0) | Corning | MT-46034CI | |
Dithiothreitol (DTT) | Sigma Aldrich | 10197777001 | Add DTT just prior to use |
Triton-X | Sigma Aldrich | T8787 | |
Nuclease-free water | Crystalgen | 221-238-10 | |
1 M Tris-HCl (pH = 7.4) | Sigma Aldrich | T2194 | |
5 M NaCl | Sigma Aldrich | 59222C | |
1 M MgCl2 | Sigma Aldrich | M1028 | |
Nonidet P40 | Sigma Aldrich | 74385 | |
Hibernate-A | Gibco | A12475-01 | |
Glutamax (100X) | Gibco | 35050-061 | |
B27 (50X) | Gibco | 17504-044 | |
1X PBS | Crystalgen | 221-133-10 | |
0.04% BSA | New England Biolabs | B9000S | |
0.2 U/μL RNAse Inhibitor | Lucigen | 30281-1 | |
Oak Ridge Centrifuge Tube | Thermo Scientific | 3118-0050 | |
Disposable Cotton-Plugged Borosilicate-Glass Pasteur Pipets | Fisher Scientific | 13-678-8B | |
Glass Tissue Dounce (2 ml) | Kimble | 885303-002 | |
Glass large clearance pestle | Kimble | 885301-0002 | |
Glass small clearance pestle | Kimble | 885302-002 | |
T 10 Basic Ultra Turrax Homogenizer | IKA | 3737001 | |
Dispersing tool (S 10 N – 5G) | IKA | 3304000 | |
Trypan Blue Stain (0.4%) | Thermo Fisher Scientific | T10282 | |
40 μm cell strainer | Falcon | 352340 | |
MACS SmartStrainers, 30 μm | Miltenyi Biotec | 130-098-458 | |
Conical tubes | Denville Scientific | 1000799 | |
Sorvall Legend XTR Centrifuge | Thermo Fisher Scientific | 75004505 | |
Fiberlite F15-6 x 100y Fixed-Angle Rotor | Thermo Fisher Scientific | 75003698 | |
Sterological Pipettes: 5 ml, 10 ml | Denville Scientific | P7127 | |
Hemocytometer | Daigger Scientific | EF16034F | |
Chemgenes Barcoding Beads | Chemgenes | Macosko-2011-10 | |
RNaseZap RNase Decontamination Solution | Invitrogen | AM9780 | |
Falcon Test Tube with Cell Strainer Cap (35 μm) | Corning | 352235 | |
MoFlo Astrios Cell Sorter | Beckman Coulter | B25982 | |
Chromium i7 Multiplex Kit, 96 rxns | 10X Genomics | 120262 | |
Chromium Single Cell 3’ Library and Gel Bead Kit v2, 4 rxns | 10X Genomics | 120267 | |
Chromium Single Cell A Chip Kit, 16 rxns | 10X Genomics | ||
Tissue Culture Dish (60 x 15 mm) | Corning | 353002 |