Name: isolation of adult spinal cord nuclei for massively parallel single-nucleus rna sequencing
Uploaded: 2018-10-12T14:30:00.000+00:00
Duration: 6 min 38 s
Description: Watch this Scientific Journal Video about Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing at JoVE.com

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.3,4,16 Given the drawbacks associated with single cell preparations, we and others have used nuclei as an alternative.16,17,18,24 This method can also be used on human tissue, including frozen spinal cord tissue.4,19,24 Here, we will describe the strengths and limitations of this approach.
Strengths of this method include the avoidance of experimentally-induced IEGs as well as the ability to use both fresh and frozen tissue.4 Thus, this approach can be useful for probing endogenous IEGs following a behavior or stimulus.1,3,4 One of the benefits of this method is that it does not require specialized devices for utilization of nuclei for massively parallel single nucleus sequencing, but can use the platform developed by Macosko et al. 2015, with minor adjustments of lysis buffer and flow rate, or use commercially available systems. Moreover, single nucleus sequencing is proven to be a comparable method to that of single cell sequencing for the identification of cell types, lending to the strength of this approach.28,29 However, there are several important limitations of this approach. Nuclei contain approximately 20-50% of cellular mRNA,29 and this is reflected in a lower number of transcripts per nucleus compared to single cell sequencing.18,29 Including intronic reads from snRNA-Seq is one approach to increase the number of detected genes.
There are several available protocols that enable isolation of nuclei from tissue.2,16,17,18,24,30 In comparison with most other methods, the protocols presented here do not require myelin removal, ultracentrifugation, or many centrifugation steps or washes that can lead to lower final numbers of nuclei. Furthermore, this protocol takes 45 min (detergent-mechanical) or 1 hour (hypotonic-mechanical) to complete. Commercial protocols supported on microfluidic platforms are more than double the time, and require many more centrifugation steps, increasing the risk of losing nuclei. In contrast with nuclei isolation protocols that involve only lysis and filtering, the methods presented here include a sucrose gradient to increase the purity of the final nuclei. This step is required for adult spinal cord tissue due to the large percentage of white matter and the resulting myelin debris.
The detergent-mechanical lysis protocol can be used for the complete tissue dissociation and lysis, and the hypotonic-mechanical lysis protocol can be used to control the amount of tissue dissociation and cellular debris allowed in the downstream application. These protocols can be used for bio-bank material, difficult to dissociate tissues and for the investigation of activity-dependent transcriptional changes through the isolation of nuclei for downstream massively parallel snRNA-Seq. In addition to massively parallel single nucleus RNA sequencing, this protocol may be used to isolate nuclei for alternative applications, including immunofluorescence and FACS and epigenetic analysis such as DNA methylation studies and ChIP-Seq (Figure 4).23

An erratum was issued for: <a href="https://www-jove-com.remotexs.ntu.edu.sg/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://www-jove-com.remotexs.ntu.edu.sg/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>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>C1016-100G</td><td></td></tr><tr><td>MgAc</td><td>Sigma Aldrich</td><td>M5661-50G</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 MgCl&lt;sub&gt;2&lt;/sub&gt;</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/&amp;mu;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 &amp;ndash; 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 &amp;mu;m cell strainer</td><td>Falcon</td><td>352340</td><td></td></tr><tr><td>MACS SmartStrainers, 30 &amp;mu;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 &amp;mu;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&amp;rsquo; 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 mm 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, Table 2). After fifteen minutes, the hypotonic-mechanical lysis yielded a crude nuclei preparation that had less debris, but also fewer nuclei (Figure 2B, Table 2). Both preparations underwent homogenization (Figure 2C and D) and sucrose density gradient centrifugation before resuspension in PBS with 0.04% BSA (Figure 2E and F). On an average, a mouse lumbar spinal cord (325.5 mg &#177; 63.9 mg standard error of the mean, SEM, N = 4) yielded 5.1 x 105 nuclei (&#177; 6.3 x 104 SEM, N = 3) following the detergent-mechanical lysis and 2.0 x 105 nuclei (&#177; 5.9 x 104 SEM, N = 3) following the hypotonic-mechanical lysis. The number of nuclei in the lumbar spinal cord was estimated from the initial crude preparation after Dounce homogenization in the detergent-mechanical lysis protocol (2.6 x 106 nuclei &#177; 4.0 x 105 SEM, N = 3, Table 2). The final sample from the detergent-mechanical lysis protocol consists of 20% of the initial nuclei (&#177; 2% SEM, N = 3, Table 2). The crude nuclei preparation from the hypotonic-mechanical lysis following trituration contains 62% of the initial nuclei (&#177; 2% SEM, N = 3, Table 2). The final hypotonic-mechanical lysis sample contains only 8% of initial nuclei (&#177; 1% SEM, N = 3, Table 2). We did not detect any difference in the total RNA yield or the cDNA yield for a housekeeping gene (Gapdh) between the two preparation methods. Using qPCR, the detergent method yielded 463.7 ng (&#177; 98.9 SEM, N= 6) of total RNA and an average detection threshold cycle of 25.2 (&#177; 1.3 SEM) for Gapdh cDNA by qPCR and the hypotonic method yielded 419.2 ng (&#177; 85.3 SEM, N = 6) of total RNA and an average detection threshold cycle of 26.1 for Gapdh cDNA (&#177; 0.8 SEM). The two lysis options both isolate nuclei from difficult-to-dissociate tissues and provide the high-quality material for the downstream single-nucleus RNA sequencing.
Given the size of microfluidic channels for downstream massively parallel single nucleus sequencing platforms, it is critical to input a nuclei suspension free of large particles or cellular debris to prevent clogging. Following the protocol presented here, there were no instances of clogging on the platform adapted from Macosko et al. 2015 (N = 17) and one partial clog on the commercial platform (N = 16).
The detergent-mechanical and hypotonic-mechanical procedures were used to isolate nuclei successfully for two massively parallel droplet encapsulation platforms and representative results are shown in Figure 3. Both of these approaches enabled transcriptional profiling of thousands of nuclei, and classification of cell types in the adult mouse lumbar spinal cord (Figure 3).4 These approaches resulted in comparable genes per nucleus for each cell type (Figure 3C and D). The rates of recovery of input nuclei between the two platforms differ. The platform adapted from Macosko et al. 2015 with modifications from Sathyamurthy et al. 2018 recovered an estimated 0.59% of nuclei (&#177; 0.05% SEM, N = 17), while the commercial platform recovered an estimated 53.7% nuclei (N = 2).
This protocol slightly enriches for neuronal nuclei in the final preparation. In lumbar spinal cord tissue sections, we found that 27% of nuclei were positive for the neuronal marker NeuN (N = 7,368 nuclei from 2 animals), while detergent-mechanical nuclei preparation of the lumbar spinal cord resulted in 31.9% of total nuclei expressing NeuN, as determined by fluorescence-activated cell sorting (FACS, &#177; 2.0% SEM, N = 13 independent nuclei preparations using pooled samples from multiple animals in each preparation, Figure 4). This is similar to what has been observed previously for the percent of NeuN-positive nuclei in the entire spinal cord (20% to 24% depending on age),27 including the cervical and thoracic regions that have more white matter and oligodendrocytes. Of note, NeuN/Rbfox3 is not expressed in all neurons and, accordingly, these numbers are likely modest underestimates. It is possible that smaller non-neuronal cells are slightly depleted during the sucrose gradient purification. In addition, downstream filtering and analysis parameters following sequencing may alter the final cell-type distribution because neurons have more genes per nucleus (Figure 3C and D) and, therefore, are less likely to be removed during the filtering process.
There are several key steps in this protocol that require care. First, excessive douncing or trituration (in steps 3 or 4, respectively) can lead to an increase in cellular debris and particle formation. Although filtration and sucrose density centrifugation can separate large particles, once small particles are generated during cellular lysis, they are difficult to remove. Secondly, during homogenization, do not place the homogenizer directly onto the bottom of the Oak Ridge tube. Instead, submerge the end of the homogenizer into the low sucrose solution containing resuspended nuclei, without touching the bottom of the tube. Homogenization improves nuclear isolation by removing cellular debris and reducing clumps and multiplets (Figure 5). Following sucrose density centrifugation, it is critical to immediately remove the Oak Ridge tube from the centrifuge, and quickly decant the supernatant in a rapid &#39;flicking&#39; motion. When resuspending nuclei from the wall of the Oak Ridge tube, resuspend the &#39;salty&#39; pellet from halfway between the myelin band and the bottom of the tube. Note that the pellet may not be visible. Resuspending nuclei higher along the tube may result in myelin contamination in the nuclei preparation. The cellular lysis and sucrose density centrifugation steps are the most critical to reducing particulates that may clog microfluidic channels for downstream application.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Stock Concentration</td>
			<td>Final Concentration</td>
			<td>Volume / Amount</td>
		</tr>
		<tr>
			<td>Detergent Lysis Buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Low sucrose buffer</td>
			<td>-</td>
			<td>-</td>
			<td>600 &#956;L</td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td>20%</td>
			<td>0.10%</td>
			<td>3 &#956;L</td>
		</tr>
		<tr>
			<td>Hypotonic Lysis buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl (pH = 7.4)</td>
			<td>1 M</td>
			<td>10 mM</td>
			<td>100 &#956;L</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>5 M</td>
			<td>10 mM</td>
			<td>20 &#956;L</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>1 M</td>
			<td>3 mM</td>
			<td>30 &#956;L</td>
		</tr>
		<tr>
			<td>Nonidet P40</td>
			<td>-</td>
			<td>0.01%</td>
			<td>1 &#956;L</td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td></td>
			<td></td>
			<td>up to 10 mL</td>
		</tr>
		<tr>
			<td>HEB Medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hibernate-A</td>
			<td>-</td>
			<td>-</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>-</td>
			<td>-</td>
			<td>100 &#956;L</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>-</td>
			<td>-</td>
			<td>200 &#956;L</td>
		</tr>
		<tr>
			<td>Low sucrose buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>-</td>
			<td>0.32 M</td>
			<td>2.75 g</td>
		</tr>
		<tr>
			<td>HEPES (pH = 8.0)</td>
			<td>1 M</td>
			<td>10 mM</td>
			<td>250 &#956;L</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>1 M</td>
			<td>5 mM</td>
			<td>125 &#956;L</td>
		</tr>
		<tr>
			<td>MgAc</td>
			<td>1 M</td>
			<td>3 mM</td>
			<td>75 &#956;L</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>0.5 M</td>
			<td>0.1 mM</td>
			<td>5 &#956;L</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>1 M</td>
			<td>1 mM</td>
			<td>25 &#956;L</td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td></td>
			<td></td>
			<td>up to 25 mL</td>
		</tr>
		<tr>
			<td>Sucrose density buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>-</td>
			<td>1 M</td>
			<td>8.6 g</td>
		</tr>
		<tr>
			<td>HEPES (pH = 8.0)</td>
			<td>1 M</td>
			<td>10 mM</td>
			<td>250 &#956;L</td>
		</tr>
		<tr>
			<td>MgAc</td>
			<td>1 M</td>
			<td>3 mM</td>
			<td>75 &#956;L</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>1 M</td>
			<td>1 mM</td>
			<td>25 &#956;L</td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td></td>
			<td></td>
			<td>up to 25 mL</td>
		</tr>
		<tr>
			<td>Resuspension Solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1X PBS</td>
			<td>-</td>
			<td>-</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>20 mg/mL</td>
			<td>0.4 mg/mL</td>
			<td>20 &#956;L</td>
		</tr>
		<tr>
			<td>RNAse Inhibitor</td>
			<td>40 U/&#956;L</td>
			<td>0.2 U/&#956;L</td>
			<td>5 &#956;L</td>
		</tr>
	</tbody>
</table>
Table 1: Table of Solutions.
<table border="0" cellpadding="0" cellspacing="0" width="453">
	<colgroup>
		<col />
		<col span="3" />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:189px;"></td>
			<td style="width:88px;">Crude</td>
			<td style="width:88px;">Homogenized</td>
			<td style="width:88px;">Final</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Detergent-Mechanical</td>
			<td>100 &#177; 15%</td>
			<td>87 &#177; 9%</td>
			<td>20 &#177; 2%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Hypotonic-Mechanical</td>
			<td>62 &#177; 12%</td>
			<td>35 &#177; 4%</td>
			<td>8 &#177; 1%</td>
		</tr>
	</tbody>
</table>
Table 2: Yield of nuclei at each step in the protocol. The number of nuclei in the initial crude preparation after dounce homogenization in the detergent-mechanical lysis protocol was used to estimate the number of nuclei in the lumbar spinal cord. The initial nuclei yield (2.6 x 106 nuclei &#177; 4.0 x 105 SEM, N = 3) was used to calculate the nuclei yield at each downstream step for both detergent- and hypotonic-mechanical lysis protocols. The number of nuclei isolated by the hypotonic-mechanical preparation was normalized to the estimated initial nuclei. Values in the table are mean &#177; SEM, N = 3.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58413/58413fig1.jpg" /> 
Figure 1: Schematic of nuclear isolation. Nuclei from the adult spinal cord can be isolated using detergent-mechanical or hypotonic-mechanical cell lysis, followed by homogenization, and sucrose density gradient centrifugation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58413/58413fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58413/58413fig2.jpg" /> 
Figure 2: Representative brightfield and DAPI-stained nuclei at key steps in the protocol.&#160;(A,B)&#160;Crude nuclei following detergent-mechanical or hypotonic-mechanical lysis. (C,D)&#160;Nuclei following homogenization. (E,F)&#160;Nuclei resuspended in PBS with 0.04% BSA following sucrose density centrifugation. Nuclei were fixed with 2% paraformaldehyde and subsequently stained using Trypan Blue or DAPI. Images were taken at 10X (Scale bar = 100 &#181;m) using brightfield and epi-fluorescence. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58413/58413fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58413/58413fig3.jpg" /> 
Figure 3: Representative tSNE plot of sequenced nuclei: using detergent-mechanical and hypotonic-mechanical lysis. (A)&#160;Results obtained from sequencing over 17,000 nuclei from the dissected adult mouse lumbar spinal cord following detergent-mechanical lysis and according to Macosko et al. 2015 with modifications from Sathyamurthy et al. 2018. This figure has been modified with permission from Sathyamurthy et al. 2018.4&#160;(B)&#160;Results obtained from sequencing 5,000 nuclei from the ejected adult lumbar spinal cord following hypotonic-mechanical lysis and a commercial microfluidic single cell encapsulation platform.26&#160;(C,D)&#160;Average genes per nucleus results following clustering of major cell types in the adult mouse spinal cord &#177; SEM. Of note, the detergent-mechanical lysis procedure followed by the Macosko et al. 2015 platform was performed using dissected lumbar spinal cord, while the hypotonic-mechanical lysis followed by the commercial platform was performed using ejected lumbar spinal cord (as described in this protocol). Given that ejecting the cord removes the dura and dorsal root ganglia, the meningeal/Schwann cell cluster is absent from Figure 3B and D. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58413/58413fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58413/58413fig4v2.jpg" /> 
Figure 4: FACS plot of NeuN+ nuclei following detergent-mechanical lysis. FACS plot showing fixed nuclei stained for NeuN (average 31.9% of total nuclei &#177; 2.0% SEM, N = 13), isolated using the detergent-mechanical lysis protocol. For the immediate fixation, nuclei for FACS validation, a crude nuclei preparation was obtained by dounce homogenization of spinal cords using the detergent-mechanical preparation, followed by immediate fixation with 1% PFA with a 5 min incubation period. Fixation was quenched with 250 mM glycine, and nuclei were collected. Staining with anti-NeuN antibody was performed in solution. FACS was performed on fixed, NeuN stained nuclei using a cell sorter. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58413/58413fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58413/58413fig5.jpg" /> 
Figure 5: Nuclei preparation without homogenization. Nuclei were resuspended prior to sucrose density centrifugation following A detergent-mechanical or B hypotonic-mechanical lysis, without homogenization. * Denotes cellular debris attached to nuclei (A) and a multiplet of nuclei attached by cellular debris (B). Nuclei resuspended in PBS with 0.04% BSA following sucrose density centrifugation. Nuclei were fixed with 2% paraformaldehyde and subsequently stained using Trypan Blue or DAPI. Images were taken at 10X (scale bar 100 &#181;m) using brightfield and epi-fluorescence. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58413/58413fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

18.6K Views.  National Institute of Neurological Disorders and Stroke. Single-cell RNA sequencing is een krachtig hulpmiddel voor het bestuderen van transcriptieprofielen van cellen, met name in heterogeen weefsel, zoals het centrale zenuwstelsel. Dit protocol biedt een methode voor het isoleren van kernen voor single-nucleus RNA sequencing. Door het gebruik van kernen in plaats van cellen, kan deze methode worden toegepast op moeilijk te scheiden weefsels, evenals bevroren weefsel.Bovendien kan deze methode worden toegepast om celtypespecifieke veranderi...

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

Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq

Adult diffuse gliomas exhibit inter- and intra-tumor heterogeneity. Until recently, the majority of large-scale molecular profiling efforts have focused on bulk approaches that led to the molecular classification of brain tumors. Over the last five years, single cell sequencing approaches have highlighted several important features of gliomas. The majority of these studies have utilized fresh brain tumor specimens to isolate single cells using flow cytometry or antibody-based separation methods. Moving forward, the use of fresh-frozen tissue samples from biobanks will provide greater flexibility to single cell applications. Furthermore, as the single-cell field advances, the next challenge will be to generate multi-omics data from either a single cell or the same sample preparation to better unravel tumor complexity. Therefore, simple and flexible protocols that allow data generation for various methods such as single-nucleus RNA sequencing (snRNA-seq) and single nucleus Assay for Transposase-Accessible Chromatin with high-throughput sequencing (snATAC-seq) will be important for the field.
Recent advances in the single cell field coupled with accessible microfluidic instruments such as the 10x genomics platform have facilitated single cell applications in research laboratories. To study brain tumor heterogeneity, we developed an enhanced protocol for the isolation of single nuclei from fresh frozen gliomas. This protocol merges existing single cell protocols and combines a homogenization step followed by filtration and buffer mediated gradient centrifugation. The resulting samples are pure single nuclei suspensions that can be used to generate single nucleus gene expression and chromatin accessibility data from the same nuclei preparation.

Intra-tumoral heterogeneity is an inherent feature of tumors, including gliomas. We developed a simple and efficient protocol that utilizes a combination of buffers and gradient centrifugation to isolate single nuclei from fresh frozen glioma tissues for single nucleus RNA and ATAC sequencing studies.

Adult diffuse gliomas exhibit inter- and intra-tumor heterogeneity. Until recently, the majority of large-scale molecular profiling efforts have focused ...

Cancer Research

Isolation of Adipose Tissue Nuclei for Single-Cell Genomic Applications

Brown and beige fat are specialized adipose tissues that dissipate energy for thermogenesis by UCP1 (Uncoupling Protein-1)-dependent and independent pathways. Until recently, thermogenic adipocytes were considered a homogeneous population. However, recent studies have indicated that there are multiple subtypes or subpopulations that are distinct in developmental origin, substrate use, and transcriptome. Despite advances in single-cell genomics, unbiased decomposition of adipose tissues into cellular subtypes has been challenging because of the fragile nature of lipid-filled adipocytes. The protocol presented was developed to circumvent these obstacles by effective isolation of single nuclei from adipose tissue for downstream applications, including RNA sequencing. Cellular heterogeneity can then be analyzed by RNA sequencing and bioinformatic analyses.

This publication describes a protocol for the isolation of nuclei from mature adipocytes, purification by fluorescence-activated sorting, and single-cell level transcriptomics.

Brown and beige fat are specialized adipose tissues that dissipate energy for thermogenesis by UCP1 (Uncoupling Protein-1)-dependent and independent ...

Genetics

Nuclei Isolation from Mouse Cardiac Progenitor Cells at Single-Cell Resolution

Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene expression and chromatin state of individual cells allows the identification of the cell type and state. Single-cell sequencing approaches have revealed a number of important characteristics of cardiac progenitor cell heterogeneity. However, these methods are generally restricted to fresh tissue, which limits studies with diverse experimental conditions, as the fresh tissue must be processed at once in the same run to reduce the technical variability. Therefore, easy and flexible procedures to produce data from methods such as single-nucleus RNA sequencing (snRNA-seq) and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing (snATAC-seq) are needed in this area. Here, we present a protocol to rapidly isolate nuclei for subsequent single-nuclei dual-omics (combined snRNA-seq and snATAC-seq). This method allows the isolation of nuclei from frozen samples of cardiac progenitor cells and can be combined with platforms that use microfluidic chambers.

Author Spotlight: Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution  

Here, we present a protocol describing cell nuclei preparation. After microdissection and enzymatic dissociation of cardiac tissue into single cells, the progenitor cells were frozen, followed by isolation of pure viable cells, which were used for single-nucleus RNA sequencing and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing analyses.

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene ...

Developmental Biology

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 ...

Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular characterization. Fluorescence-activated nuclei sorting (FANS) can be used to successfully isolate and study human neuronal nuclei (NeuN+) populations from frozen archival tissue, thereby avoiding problems associated with handling&#160;fresh tissue. However, efforts to similarly isolate astroglia from the non-neuronal (NeuN-) element are lacking. A recently developed and validated immunotagging strategy uses three transcription factor antibodies to simultaneously isolate enriched neuronal (NeuN+), astrocyte (paired box protein 6 (PAX6)+NeuN-), and oligodendrocyte progenitor (OLIG2+NeuN-) nuclei populations from non-diseased, fresh (unfixed) snap-frozen&#160;postmortem human temporal neocortex tissue.
This technique was shown to be useful for the characterization of cell type-specific transcriptome alterations in primary pathological epilepsy neocortex. Transcriptomic analyses confirmed that PAX6+NeuN- sorted populations are robustly enriched for pan-astrocyte markers and capture astrocytes in both resting and reactive conditions. This paper describes the FANS methodology for the isolation of astrocyte-enriched nuclei populations from fresh-frozen human cortex, including tissue dissociation into single-nucleus (sn) suspension; immunotagging of nuclei with anti-NeuN and anti-PAX6 fluorescently conjugated antibodies; FANS gating strategies and quality control metrics for optimizing sensitivity and specificity during sorting and for confirming astrocyte enrichment; and recommended procurement for downstream transcriptome and chromatin accessibility sequencing at bulk or sn resolution. This protocol is applicable for non-necrotic, fresh-frozen, human cortical specimens with various pathologies and recommended postmortem tissue collection within 24 h.

We have developed a method that enriches for and isolates human astrocyte populations from fresh-frozen tissue for use in downstream molecular analyses.

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular ...

Low-input Nucleus Isolation and Multiplexing with Barcoded Antibodies of Mouse Sympathetic Ganglia for Single-nucleus RNA Sequencing

The cardiac autonomic nervous system is crucial in controlling cardiac function, such as heart rate and cardiac contractility, and is divided into sympathetic and parasympathetic branches. Normally, there is a balance between these two branches to maintain homeostasis. However, cardiac disease states such as myocardial infarction, heart failure, and hypertension can induce the remodeling of cells involved in cardiac innervation, which is associated with an adverse clinical outcome.
Although there are vast amounts of data for the histological structure and function of the cardiac autonomic nervous system, its molecular biological architecture in health and disease is still enigmatic in many aspects. Novel technologies such as single-cell RNA sequencing (scRNA-seq) hold promise for the genetic characterization of tissues at single-cell resolution. However, the relatively large size of neurons may impede the standardized use of these techniques. Here, this protocol exploits droplet-based single-nucleus RNA sequencing (snRNA-seq), a method to characterize the biological architecture of cardiac sympathetic neurons in health and disease. A stepwise approach is demonstrated to perform snRNA-seq of the bilateral superior cervical (SCG) and stellate ganglia (StG) dissected from adult mice.
This method enables long-term sample preservation, maintaining an adequate RNA quality when samples from multiple individuals/experiments cannot be collected all at once within a short period of time. Barcoding the nuclei with hashtag oligos (HTOs) enables demultiplexing and the trace-back of distinct ganglionic samples post sequencing. Subsequent analyses revealed successful nuclei capture of neuronal, satellite glial, and endothelial cells of the sympathetic ganglia, as validated by snRNA-seq. In summary, this protocol provides a stepwise approach for snRNA-seq of sympathetic extrinsic cardiac ganglia, a method that has the potential for broader application in studies of the innervation of other organs and tissues.

This protocol describes the detailed, low-input sample preparation for single-nucleus sequencing, including the dissection of mouse superior cervical and stellate ganglia, cell dissociation, cryopreservation, nucleus isolation, and hashtag barcoding.

The cardiac autonomic nervous system is crucial in controlling cardiac function, such as heart rate and cardiac contractility, and is divided into ...

Medicine

Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension with&#160;intact cell or nuclei involves dissociation and permeabilization, steps that can introduce unwanted noise and undesirable damage. Particularly, certain cell-types such as neurons are challenging to dissociate into individual cells. Additionally, permeabilization of the cellular membrane to release nuclei requires optimization by trial-and-error, which can be time consuming, labor intensive and financially nonviable. To enhance the robustness and reproducibility of sample preparation for high-throughput sequencing, we describe a rapid enzyme and detergent-free column-based nuclei isolation method. The protocol enables efficient isolation of nuclei from the entire zebrafish brain within 20 minutes. The isolated nuclei display intact nuclear morphology and low propensity to aggregate. Further, flow cytometry allows nuclei enrichment and clearance of cellular debris for downstream application. The protocol, which should work on soft tissues and cultured cells, provides a simple and accessible method for sample preparation that can be utilized for high-throughput profiling, simplifying the steps required for successful single-nuclei RNA-seq and ATAC-seq experiments.

Single nuclei isolation relies on dissociation and detergent-based permeabilization of the cell membrane, steps that need optimization and are prone to introducing technical artifacts. We demonstrate a detergent and enzyme-free protocol for rapid isolation of intact nuclei directly from whole tissue, yielding nuclei suitable for single-nucleus RNA-seq (snRNA-Seq) or ATAC-seq.

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension ...

Biology

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are ...

Isolation of Nuclei from Flash-Frozen Liver Tissue for Single-Cell Multiomics

The liver is a complex and heterogenous tissue responsible for carrying out many critical physiological functions, such as the maintenance of energy homeostasis and the metabolism of xenobiotics, among others. These tasks are performed through tight coordination between hepatic parenchymal and non-parenchymal cells. Additionally, various metabolic activities are confined to specific areas of the hepatic lobule-a phenomenon called liver zonation. Recent advances in single-cell sequencing technologies have empowered researchers to investigate tissue heterogeneity at a single-cell resolution. In many complex tissues, including the liver, harsh enzymatic and/or mechanical dissociation protocols can negatively affect the viability or the quality of the single-cell suspensions needed to comprehensively characterize this organ in health and disease.
This paper describes a robust and reproducible protocol for isolating nuclei from frozen, archived liver tissues. This method yields high-quality nuclei that are compatible with downstream, single-cell omics approaches, including single-nucleus RNA-seq, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), as well as multimodal omics (joint RNA-seq and ATAC-seq). This method has been successfully used for the isolation of nuclei from healthy and diseased human, mouse, and non-human primate frozen liver samples. This approach allows the unbiased isolation of all the major cell types in the liver and, therefore, offers a robust methodology for studying the liver at the single-cell resolution.

Here, we present a protocol for isolating nuclei from flash-frozen, archived liver tissues for single-nucleus RNA-seq, ATAC-seq, and joint multiomics (RNA-seq and ATAC-seq).

The liver is a complex and heterogenous tissue responsible for carrying out many critical physiological functions, such as the maintenance of energy ...

Isolation and Profiling of Single Nuclei from Frozen Mammalian Tissues

A Simple, Quick, and Partially Automated Protocol for the Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nucleus Sequencing

Single-cell and single-nucleus RNA sequencing have become common laboratory applications due to the wealth of transcriptomic information that they provide. Single nucleus RNA sequencing, particularly, is useful for investigating gene expression in difficult-to-dissociate tissues. Furthermore, this approach is also compatible with frozen (archival) material. Here, we describe a protocol to isolate high-quality single nuclei from frozen mammalian tissues for downstream single nucleus RNA sequencing in a partially-automated manner using commercially available instruments and reagents. Specifically, a robotic dissociator is used to automate and standardize tissue homogenization, followed by an optimized chemical gradient to filter the nuclei. Lastly, we accurately and automatically count the nuclei using an automated fluorescent cell counter. The performance of this protocol is demonstrated on mouse brain, rat kidney, and cynomolgus liver and spleen tissue. This protocol is straightforward, rapid, and readily adaptable to various mammalian tissues without requiring extensive optimization and provides good quality nuclei for downstream single nuclei RNA sequencing.

Author Spotlight: Enhancing Drug Discovery - Development of Automated, Standardized Protocols for Nuclei Extraction from Frozen Tissues 

The study describes a simple, quick, and partially automated protocol to isolate high-quality nuclei from frozen mammalian tissues for downstream single nuclei RNA sequencing.

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

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