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

Summary

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.

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.

Introduction

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.

Protocol

All animal work was performed in accordance with a protocol approved by the National Institute of Neurological Disorders and Stroke Animal Care and Use Committee. Balanced samples of male and female ICR/CD-1 wild-type mice, between 8 and 12 weeks old, were used for all experiments. Mice should be handled in accordance with local Institutional Animal Care and Use Committee guidelines.

1. Preparation of Materials and Buffers

1. Prepare all buffers the day of use and pre-chill on ice (see Table 1).
   1. If using detergent-mechanical lysis, prepare the detergent lysis buffer (> 500 μL per sample), low sucrose buffer (> 6 mL per sample), sucrose density buffer (> 12.5 mL per sample), and the resuspension solution (> 1 mL).
   2. If using hypotonic-mechanical lysis, prepare the hypotonic lysis buffer (> 5 mL per sample), HEB medium (> 5 mL per sample), low sucrose buffer (> 3 mL per sample), sucrose density buffer (>12.5 mL per sample), and the resuspension solution (> 1 mL).
   3. Add 25 μL of dithiothreitol (DTT) to 25 mL of the low sucrose buffer and another 25 μL of DTT to 25 mL of the sucrose density gradient buffer just before starting the protocol.
2. Cover the dissecting surface with aluminum-foil to minimize contamination of the sample with fibers from paper towels or bench protectors, which can clog microfluidic channels used for capturing single nuclei.
3. Spray dissecting tools and bench space with an RNase decontamination solution. Additionally, spray the inside of the Dounce homogenizer tube (if using detergent-mechanical cell lysis) and Oak Ridge tube with an RNase decontamination solution. Rinse out the Dounce and Oak Ridge tube with ultrapure, RNase-free water.
4. Pre-chill all collection tubes (50 mL conical, Oak Ridge) and Dounce homogenizer tubes on ice.
5. Fire polish a series of Pasteur pipettes (if using hypotonic-mechanical cell lysis).

2. Preparation of the Spinal Cord

1. If using fresh tissue, euthanize the mouse by CO₂ inhalation. Following euthanasia, spray the coat of the mouse with 70% ethanol to minimize hair contamination in the sample.
2. Decapitate the mouse with sharp, RNase-free surgical scissors. Next, gently lifting the abdominal skin with forceps and make an incision along the length of the body to expose the inner organs.
3. Eviscerate the mouse by pulling the inner organs from the body cavity using forceps. Do not use paper towels to clean the area or to remove organs as this may introduce contaminants. Using scissors, cut the vertebral column between the L2 and L3 spinal vertebrae.
   NOTE: With practice, this step can be achieved in less than 30 seconds.
   1. To eject the spinal cord, fit a 3 mL syringe containing ice-cold PBS with a 25 G ¼ inch needle. Place the tip of the needle into the sacral end of the vertebral column. Use two fingers to pinch the vertebrae to create a tight seal around the tip of the needle and press down on the plunger to eject the spinal cord rostrally. Place the spinal cord in a petri dish with ice-cold PBS.
   2. At this point, freeze the tissue and store at -80 °C or use immediately for either detergent-mechanical (Step 3) or hypotonic-mechanical (Step 4) lysis.
4. If using frozen tissue, maintain the tissue on dry ice, proceed to detergent-mechanical (Step 3) or hypotonic-mechanical (Step 4) lysis.

3. Detergent-Mechanical Cell Lysis

1. Place the lumbar spinal cord in a pre-chilled Dounce homogenizer and add 500 μL pre-chilled detergent lysis buffer.
   NOTE: A mouse lumbar spinal cord is 325.5 mg ± 63.9 mg standard error of the mean (SEM, N = 4). 50 mg–1.5 g of tissue can be successfully used.
2. Dounce with 5 strokes of pestle A (‘loose’ pestle), then 5-10 strokes of pestle B (‘tight’ pestle). Avoid lifting the homogenizer out of the lysis solution in between strokes and avoid introducing bubbles.
3. Place a 40 μm strainer over a pre-chilled 50 mL conical tube and prewet with 1 mL of low sucrose buffer.
4. Add 1 mL of low sucrose buffer to the Dounce homogenizer containing the crude nuclei in the lysis buffer and mix gently by pipetting 2–3 times.
5. Pass the crude nuclei prep over the 40 μm strainer into the pre-chilled 50 mL conical tube.
6. Pass an additional 1 mL low sucrose buffer over the 40 μm strainer, bringing the final volume to 3 mL of the low sucrose buffer and 500 μL of the lysis buffer.
7. Repeat steps 3.1–3.6 if combining multiple cords, pooling in the same conical tube.
8. Centrifuge the sample at 3,200 x g for 10 min at 4 °C. Once the centrifugation is complete, decant the supernatant. Proceed to Step 5.

4. Hypotonic-mechanical Cell Lysis

1. Place the lumbar spinal cord in 5 mL of the hypotonic lysis buffer in a tissue culture dish. Use the blunt end of spring scissors to bisect the spinal cord, then use spring scissors to cut the cord into 3–4 mm pieces, but do not mince.
   Note: 50 mg–1.5 g of tissue can be successfully used.
2. Incubate on the ice for 15 min, swirling 2–3 times.
3. Add 5 mL of HEB medium to dilute the hypotonic lysis buffer.
4. Triturate the tissue 10 times with a 5 mL serological pipette, or until all of the pieces of the tissue move smoothly through the opening of the pipette.
5. Triturate with a series of three fire-polished Pasteur pipettes with progressively narrower diameters (~900–600 μm).
   1. For each pipette, triturate 5-15 times, allow tissue to settle, remove 1–2 mL of supernatant containing dissociated nuclei and pass over a 40 μm strainer into a pre-chilled 50 mL conical tube.
   2. After trituration with the smallest-sized Pasteur pipette, ensure that the homogenate flows smoothly through the pipette tip. Pass the remaining solution over the 40 μm strainer into the 50 mL conical tube.
      NOTE: The total number of triturations can be adjusted as desired. The meninges of the mouse spinal cord will remain, but it is important to triturate any visible chunks of spinal cord. Pass the remaining homogenate over the 40 μm strainer. Avoid introducing bubbles during trituration.
6. Centrifuge the filtered sample at 1,000 x g for 10 min at 4 °C. Once the centrifugation is complete, decant and discard the supernatant. Proceed to Step 5.

5. Homogenization and Sucrose Density Gradient

1. After either Step 3 or 4, resuspend the pellet using 3 mL of low sucrose buffer. Gently swirl to remove the pellet from the wall to facilitate the resuspension. Let the sample sit on ice for 2 min and transfer the suspension to an Oak Ridge tube.
2. Using the homogenizer at setting 1, homogenize the nuclei in low sucrose buffer for 15–30 s, keeping the sample on ice.
   NOTE: Use 15 s if using one lumbar spinal cord or 30 s if using pooled samples or a whole spinal cord.
3. Using a serological pipette, layer 12.5 mL of density sucrose buffer underneath the low sucrose buffer homogenate, taking care not to create a bubble that disrupts the density layers.
4. Centrifuge the tubes at 3,200 x g for 20 min at 4 °C.
5. Once the centrifugation is complete, immediately decant the supernatant in a flicking motion.
   NOTE: A residual volume (less than 400 μL) of sucrose buffer can be discarded if desired to produce a lower volume and cleaner final sample, but this residual volume does contain nuclei and can be preserved to maximize nuclei yield.
6. Using 100 μL - 1 mL of resuspension solution, resuspend the nuclei remaining on the wall. Avoid the myelin ‘frown’ that remains with the detergent-based preparation.
7. Filter the nuclei through a 30–35 μm pore-size strainer and collect in a pre-chilled tube.
8. Determine the nuclei yield using a hemocytometer to count nuclei under a 10X objective.
   NOTE: Trypan blue can be added to visualize nuclei, which should appear blue. Note the amount of cellular debris.
9. Proceed to either Step 6 or 7.

6. Massively Parallel snRNA-Sequencing: Academic Platform⁷

1. Perform the massively parallel snRNA sequencing (e.g., Drop-Seq) method as previously described⁷ with the following modifications:⁴
   1. Adjust nuclei to a final concentration of 225 nuclei per μL.
   2. Prepare barcoded beads at a concentration of 250 beads per μL.
   3. Prepare the lysis buffer with 0.7% sarkosyl.
   4. Adjust the flow rates to 35 μL per min for beads, 35 μL per min for nuclei, and 200 μL per min for oil.

7. Massively parallel snRNA-sequencing: Commercial Platform²⁶

1. Perform massively parallel snRNA-sequencing using the commercial platform (e.g., Chromium Single Cell Gene Expression Solution) products according to the manufacturer’s instructions²⁶ with the following modification:
   1. Following reverse-transcription, add an additional PCR cycle to the calculated number of cycles for cDNA amplification based on the targeted cell recovery to compensate for decreased cDNA from nuclei compared to cells.

Results

Here, we performed isolation of nuclei from the adult mouse lumbar spinal cord for downstream massively parallel RNA sequencing. The protocol involved three main components: tissue disruption and cellular lysis, homogenization, and sucrose density centrifugation (Figure 1). Within seconds, the detergent-mechanical lysis yielded a crude nuclei preparation with a large number of nuclei as well as cellular and tissue debris (Figure 2A

Discussion

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.

Materials

Name	Company	Catalog Number	Comments
Sucrose	Invitrogen	15503-022
1 M HEPES (pH = 8.0)	Gibco	15630-080
CaCl2	Sigma Aldrich	C1016-100G
MgAc	Sigma Aldrich	M5661-50G
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 mm x 15 mm)	Corning	353002