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.

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
<ol>
	<li>Prepare all buffers the day of use and pre-chill on ice (see Table 1).
	<ol>
		<li>If using detergent-mechanical lysis, prepare the detergent lysis buffer (&#62; 500 &#956;L per sample), low sucrose buffer (&#62; 6 mL per sample), sucrose density buffer (&#62; 12.5 mL per sample), and the resuspension solution (&#62; 1 mL).</li>
		<li>If using hypotonic-mechanical lysis, prepare the hypotonic lysis buffer (&#62; 5 mL per sample), HEB medium (&#62; 5 mL per sample), low sucrose buffer (&#62; 3 mL per sample), sucrose density buffer (&#62;12.5 mL per sample), and the resuspension solution (&#62; 1 mL).</li>
		<li>Add 25 &#956;L of dithiothreitol (DTT) to 25 mL of the low sucrose buffer and another 25 &#956;L of DTT to 25 mL of the sucrose density gradient buffer just before starting the protocol.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
	<li>Pre-chill all collection tubes (50 mL conical, Oak Ridge) and Dounce homogenizer tubes on ice.</li>
	<li>Fire polish a series of Pasteur pipettes (if using hypotonic-mechanical cell lysis).</li>
</ol>
2. Preparation of the Spinal Cord
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.
	<ol>
		<li>To eject the spinal cord, fit a 3 mL syringe containing ice-cold PBS with a 25 G &#188; 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.</li>
		<li>At this point, freeze the tissue and store at -80 &#176;C or use immediately for either detergent-mechanical (Step 3) or hypotonic-mechanical (Step 4) lysis.</li>
	</ol>
	</li>
	<li>If using frozen tissue, maintain the tissue on dry ice, proceed to detergent-mechanical (Step 3) or hypotonic-mechanical (Step 4) lysis.</li>
</ol>
3. Detergent-Mechanical Cell Lysis
<ol>
	<li>Place the lumbar spinal cord in a pre-chilled Dounce homogenizer and add 500 &#956;L pre-chilled detergent lysis buffer. 
	NOTE: A mouse lumbar spinal cord is 325.5 mg &#177; 63.9 mg standard error of the mean (SEM, N = 4). 50 mg&#8211;1.5 g of tissue can be successfully used.</li>
	<li>Dounce with 5 strokes of pestle A (&#8216;loose&#8217; pestle), then 5-10 strokes of pestle B (&#8216;tight&#8217; pestle). Avoid lifting the homogenizer out of the lysis solution in between strokes and avoid introducing bubbles.</li>
	<li>Place a 40 &#956;m strainer over a pre-chilled 50 mL conical tube and prewet with 1 mL of low sucrose buffer.</li>
	<li>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&#8211;3 times.</li>
	<li>Pass the crude nuclei prep over the 40 &#956;m strainer into the pre-chilled 50 mL conical tube.</li>
	<li>Pass an additional 1 mL low sucrose buffer over the 40 &#956;m strainer, bringing the final volume to 3 mL of the low sucrose buffer and 500 &#956;L of the lysis buffer.</li>
	<li>Repeat steps 3.1&#8211;3.6 if combining multiple cords, pooling in the same conical tube.</li>
	<li>Centrifuge the sample at 3,200 x g for 10 min at 4 &#176;C. Once the centrifugation is complete, decant the supernatant. Proceed to Step 5.</li>
</ol>
4. Hypotonic-mechanical Cell Lysis
<ol>
	<li>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&#8211;4 mm pieces, but do not mince. 
	Note: 50 mg&#8211;1.5 g of tissue can be successfully used.</li>
	<li>Incubate on the ice for 15 min, swirling 2&#8211;3 times.</li>
	<li>Add 5 mL of HEB medium to dilute the hypotonic lysis buffer.</li>
	<li>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.</li>
	<li>Triturate with a series of three fire-polished Pasteur pipettes with progressively narrower diameters (~900&#8211;600 &#956;m).
	<ol>
		<li>For each pipette, triturate 5-15 times, allow tissue to settle, remove 1&#8211;2 mL of supernatant containing dissociated nuclei and pass over a 40 &#956;m strainer into a pre-chilled 50 mL conical tube.</li>
		<li>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 &#956;m 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 &#956;m strainer. Avoid introducing bubbles during trituration.</li>
	</ol>
	</li>
	<li>Centrifuge the filtered sample at 1,000 x g for 10 min at 4 &#176;C. Once the centrifugation is complete, decant and discard the supernatant. Proceed to Step 5.</li>
</ol>
5. Homogenization and Sucrose Density Gradient
<ol>
	<li>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.</li>
	<li>Using the homogenizer at setting 1, homogenize the nuclei in low sucrose buffer for 15&#8211;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.</li>
	<li>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.</li>
	<li>Centrifuge the tubes at 3,200 x g for 20 min at 4 &#176;C.</li>
	<li>Once the centrifugation is complete, immediately decant the supernatant in a flicking motion. 
	NOTE: A residual volume (less than 400 &#956;L) 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.</li>
	<li>Using 100 &#956;L - 1 mL of resuspension solution, resuspend the nuclei remaining on the wall. Avoid the myelin &#8216;frown&#8217; that remains with the detergent-based preparation.</li>
	<li>Filter the nuclei through a 30&#8211;35 &#956;m pore-size strainer and collect in a pre-chilled tube.</li>
	<li>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.</li>
	<li>Proceed to either Step 6 or 7.</li>
</ol>
6. Massively Parallel snRNA-Sequencing: Academic Platform7
<ol>
	<li>Perform the massively parallel snRNA sequencing (e.g., Drop-Seq) method as previously described7 with the following modifications:4
	<ol>
		<li>Adjust nuclei to a final concentration of 225 nuclei per &#956;L.</li>
		<li>Prepare barcoded beads at a concentration of 250 beads per &#956;L.</li>
		<li>Prepare the lysis buffer with 0.7% sarkosyl.</li>
		<li>Adjust the flow rates to 35 &#956;L per min for beads, 35 &#956;L per min for nuclei, and 200 &#956;L per min for oil.</li>
	</ol>
	</li>
</ol>
7. Massively parallel snRNA-sequencing: Commercial Platform26
<ol>
	<li>Perform massively parallel snRNA-sequencing using the commercial platform (e.g., Chromium Single Cell Gene Expression Solution) products according to the manufacturer&#8217;s instructions26 with the following modification:
	<ol>
		<li>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.</li>
	</ol>
	</li>
</ol>

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We have no conflicts of interest to disclose.

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...

An erratum was issued for: <a href="https://jove.unicartagenaproxy.elogim.com/video/58413/isolation-adult-spinal-cord-nuclei-for-massively-parallel-single">Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing</a>. The Protocol section was updated.
Step 3.1 was updated from:
Place the lumbar spinal cord in a pre-chilled Dounce homogenizer and add 500 mL pre-chilled detergent lysis buffer.
to:
Place the lumbar spinal cord in a pre-chilled Dounce homogenizer and add 500 &#956;L pre-chilled detergent lysis buffer.
Step 3.6 was updated from:
Pass an additional 1 mL low sucrose buffer over the 40 mm strainer, bringing the final volume to 3 mL of the low sucrose buffer and 500 mL of the lysis buffer.
to:
Pass an additional 1 mL low sucrose buffer over the 40 mm strainer, bringing the final volume to 3 mL of the low sucrose buffer and 500 &#956;L of the lysis buffer.
Step 5.5 was updated from:
Once the centrifugation is complete, immediately decant the supernatant in a flicking motion. 
NOTE: A residual volume (less than 400 mL) 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
to:
Once the centrifugation is complete, immediately decant the supernatant in a flicking motion. 
NOTE: A residual volume (less than 400 &#956;L) 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
Step 5.6 was updated from:
Using 100 mL - 1 mL of resuspension solution, resuspend the nuclei remaining on the wall. Avoid the myelin &#8216;frown&#8217; that remains with the detergent-based preparation.
to:
Using 100 &#956;L - 1 mL of resuspension solution, resuspend the nuclei remaining on the wall. Avoid the myelin &#8216;frown&#8217; that remains with the detergent-based preparation.
Steps 6.1.1 - 6.1.4 were&#160;updated from:
<ol>
	<li>Adjust nuclei to a final concentration of 225 nuclei per mL.</li>
	<li>Prepare barcoded beads at a concentration of 250 beads per mL.</li>
	<li>Prepare the lysis buffer with 0.7% sarkosyl.</li>
	<li>Adjust the flow rates to 35 mL per min for beads, 35 mL per min for nuclei, and 200 mL per min for oil.</li>
</ol>
to:
<ol>
	<li>Adjust nuclei to a final concentration of 225 nuclei per &#956;L.</li>
	<li>Prepare barcoded beads at a concentration of 250 beads per &#956;L.</li>
	<li>Prepare the lysis buffer with 0.7% sarkosyl.</li>
	<li>Adjust the flow rates to 35 &#956;L per min for beads, 35 &#956;L per min for nuclei, and 200 &#956;L per min for oil.</li>
</ol>

An erratum was issued for: <a href="https://jove.unicartagenaproxy.elogim.com/video/58413/isolation-adult-spinal-cord-nuclei-for-massively-parallel-single">Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing</a>. The Protocol section was updated.

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

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&#160;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.

<table>
	<tbody>
		<tr>
			<td>Sucrose</td>
			<td>Invitrogen</td>
			<td>15503-022</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M HEPES (pH = 8.0)</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma Aldrich</td>
			<td>C1016-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>MgAc</td>
			<td>Sigma Aldrich</td>
			<td>M1028-10X1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M EDTA (pH = 8.0)</td>
			<td>Corning</td>
			<td>MT-46034CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Sigma Aldrich</td>
			<td>10197777001</td>
			<td>Add DTT just prior to use</td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td>Sigma Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>Crystalgen</td>
			<td>221-238-10</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M Tris-HCl (pH = 7.4)</td>
			<td>Sigma Aldrich</td>
			<td>T2194</td>
			<td></td>
		</tr>
		<tr>
			<td>5 M NaCl</td>
			<td>Sigma Aldrich</td>
			<td>59222C</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M MgCl2</td>
			<td>Sigma Aldrich</td>
			<td>M1028</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonidet P40</td>
			<td>Sigma Aldrich</td>
			<td>74385</td>
			<td></td>
		</tr>
		<tr>
			<td>Hibernate-A</td>
			<td>Gibco</td>
			<td>A12475-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax (100X)</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 (50X)</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>1X PBS</td>
			<td>Crystalgen</td>
			<td>221-133-10</td>
			<td></td>
		</tr>
		<tr>
			<td>0.04% BSA</td>
			<td>New England Biolabs</td>
			<td>B9000S</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 U/&#956;L RNAse Inhibitor</td>
			<td>Lucigen</td>
			<td>30281-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Oak Ridge Centrifuge Tube</td>
			<td>Thermo Scientific</td>
			<td>3118-0050</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Cotton-Plugged Borosilicate-Glass Pasteur Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-8B</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Tissue Dounce (2 ml)</td>
			<td>Kimble</td>
			<td>885303-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass large clearance pestle</td>
			<td>Kimble</td>
			<td>885301-0002</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass small clearance pestle</td>
			<td>Kimble</td>
			<td>885302-002</td>
			<td></td>
		</tr>
		<tr>
			<td>T 10 Basic Ultra Turrax Homogenizer</td>
			<td>IKA</td>
			<td>3737001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispersing tool (S 10 N &#8211; 5G)</td>
			<td>IKA</td>
			<td>3304000</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain (0.4%)</td>
			<td>Thermo Fisher Scientific</td>
			<td>T10282</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#956;m cell strainer</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS SmartStrainers, 30 &#956;m</td>
			<td>Miltenyi Biotec</td>
			<td>130-098-458</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tubes</td>
			<td>Denville Scientific</td>
			<td>1000799</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall Legend XTR Centrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>75004505</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiberlite F15-6 x 100y Fixed-Angle Rotor</td>
			<td>Thermo Fisher Scientific</td>
			<td>75003698</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterological Pipettes: 5 ml, 10 ml</td>
			<td>Denville Scientific</td>
			<td>P7127</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Daigger Scientific</td>
			<td>EF16034F</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemgenes Barcoding Beads</td>
			<td>Chemgenes</td>
			<td>Macosko-2011-10</td>
			<td></td>
		</tr>
		<tr>
			<td>RNaseZap RNase Decontamination Solution</td>
			<td>Invitrogen</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Test Tube with Cell Strainer Cap (35 &#956;m)</td>
			<td>Corning</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>MoFlo Astrios Cell Sorter</td>
			<td>Beckman Coulter</td>
			<td>B25982</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromium i7 Multiplex Kit, 96 rxns</td>
			<td>10X Genomics</td>
			<td>120262</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromium Single Cell 3&#8217; Library and Gel Bead Kit v2, 4 rxns</td>
			<td>10X Genomics</td>
			<td>120267</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromium Single Cell A Chip Kit, 16 rxns</td>
			<td>10X Genomics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Dish (60 x 15 mm)</td>
			<td>Corning</td>
			<td>353002</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of adult spinal cord nuclei for massively parallel single-nucleus rna sequencing

Probing an individual cell&#39;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.

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</stron...

Watch this Scientific Journal Video about Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing at JoVE.com

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

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&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

isolation-adult-spinal-cord-nuclei-for-massively-parallel-single

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

18.6K Views.  National Institute of Neurological Disorders and Stroke. 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.

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

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

Induction of Complete Transection-Type Spinal Cord Injury in Mice

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, such as cell transplantation methods, are being researched to overcome the debilitating disabilities arising from SCI. Most pre-clinical animal trials are conducted in rodent models of SCI. While rat models of SCI have been widely used, mouse models have received less attention, even though mouse models can have significant advantages over rat models. The small size of mice equates to lower animal maintenance costs than for rats, and the availability of numerous transgenic mouse models is advantageous for many types of studies. Inducing repeatable and precise injury in the animals is the primary challenge for SCI research, which in small rodents requires high-precision surgery. The transection-type injury model has been a commonly used injury model over the last decade for transplantation-based therapeutic research, however a standardized method for inducing a complete transection-type injury in mice does not exist. We have developed a surgical protocol for inducing a complete transection type injury in C57BL/6 mice at thoracic vertebral level 10 (T10). The procedure uses a small tip drill instead of rongeurs to precisely remove the lamina, after which a thin blade with rounded cutting edge is used to induce the spinal cord transection. This method leads to reproducible transection-type injury in small rodents with minimal collateral muscle and bone damage and therefore minimizes confounding factors, specifically where behavioral functional outcomes are analyzed.

This protocol describes how to create a precise laminectomy for induction of stable transection-type spinal cord injury in the mouse model, with minimal collateral damage for spinal cord injury research.

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, ...

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

Summary

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Chapters in this video

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Induction of Complete Transection-Type Spinal Cord Injury in Mice