Name: low-input nucleus isolation and multiplexing with barcoded antibodies of mouse sympathetic ganglia for single-nucleus rna sequencing
Uploaded: 2022-03-23T12:30:00
Description: Watch this Scientific Journal Video about Low-input Nucleus Isolation and Multiplexing with Barcoded Antibodies of Mouse Sympathetic Ganglia for Single-nucleus RNA Sequencing at JoVE.com

We thank Susan L. Kloet (Department of Human Genetics, LUMC, Leiden, the Netherlands) for her help in experimental design and useful discussions. We thank Emile J. de Meijer (Department of Human Genetics, LUMC, Leiden, the Netherlands) for the help with single-nucleus RNA isolation and library preparation for sequencing. This work is supported by the Netherlands Organization for Scientific Research (NWO) [016.196.346 to M.R.M.J.].

This protocol describes all steps required for the snRNA-seq of murine cervical and/or cervicothoracic (stellate) ganglia. Female and male C57BL/6J mice (15 weeks old, n = 2 for each sex) were used. One additional Wnt1-Cre;mT/mG mouse was used to visualize the ganglia for dissection purposes17,18. This additional mouse was generated by the crossbreeding of a B6.Cg-Tg(Wnt1-cre)2Sor/J mouse and a B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J mouse. All anim...

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Here, a detailed protocol is described that focuses on i) the dissection of adult mouse superior cervical and stellate sympathetic ganglia, ii) the isolation and cryopreservation of the ganglionic cells, iii) nucleus isolation, and iv) nucleus-barcoding with HTO labeling for multiplexing purposes and snRNA-seq.
With this protocol, sympathetic ganglionic cells can easily be obtained by dissociating individual ganglia using commonly used trypsin and collagenase. Long-term preservation of isolate...

The autonomic nervous system (ANS) is a crucial part of the peripheral nervous system that maintains body homeostasis, including the adaption to environmental conditions and pathology1. It is involved in the regulation of multiple organ systems throughout the body such as the cardiovascular, respiratory, digestive, and endocrine systems. The ANS is divided into sympathetic and parasympathetic branches. Spinal branches of the sympathetic nervous system synapse in ganglia of the sympathetic chain, situated bilaterally in a paravertebral position. The bilateral cervical and thoracic ganglia, especially the StG, are important components participating in cardiac sympathetic innervation. In disease states, such as cardiac ischemia, neuronal remodeling can occur, resulting in a sympathetic overdrive2. The neuronal remodeling has been demonstrated in multiple histological studies in humans and several other animal species3,4,5,6. A detailed biological characterization of cardiac ischemia-induced neuronal remodeling in cardiac sympathetic ganglia is currently lacking, and the fundamental biological characteristics of specialized neuronal cell types or subtypes within the cardiac sympathetic nervous system (SNS) are not fully determined yet in health and disease7.
Novel technologies, such as scRNA-seq, have opened gateways for the genetic characterization of small tissues on a single-cell level8,9. However, the relatively large size of neurons may impede the optimized use of these single-cell techniques in humans10. In addition, single-cell sequencing requires a high-throughput of cells to recover a sufficient cell number due to a high loss in the sequencing process. This might prove challenging when studying small tissues that are hard to capture in one session and require multiple samples to introduce enough single cells for sequencing. The recently developed droplet-based snRNA-seq technology (i.e., the 10x Chromium platform) allows the study of biological differences among single nuclei11,12. snRNA-seq holds an advantage over scRNA-seq for large cells (&#62;30 &#181;m), which may not be captured in Gel Bead in Emulsions (GEMs), as well as improved compatibility with extensive dissociation and/or prolonged preservation13,14,15.
Heterogeneity, the number of neuronal cells, and other cells enriched in the cardiac SNS are important aspects for the characterization of the ANS in health and disease states. In addition, the organ- or region-specific innervation by each sympathetic ganglion contributes to the complexity of the SNS. Moreover, cervical, stellate, and thoracic ganglia of the sympathetic chain have been shown to innervate different regions of the heart16. Therefore, it is necessary to perform single-nucleus analysis of ganglionic cells derived from individual ganglia to study their biological architecture.
Droplet-based snRNA-seq allows transcriptome-wide expression profiling for a pool of thousands of cells from multiple samples at once with lower costs than plate-based sequencing platforms. This approach enables droplet-based snRNA-seq to be more suitable for cellular phenotype classification and new subpopulation identification of cells within the SCG and the StG. Notably, this protocol provides a concise stepwise approach for the identification, isolation, and single-nucleus RNA sequencing of sympathetic extrinsic cardiac ganglia, a method that has the potential for a broad application in studies of the characterization of ganglia innervating other related organs and tissues in health and disease.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemicals and reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>0.4% trypan blue dye</td>
			<td>Bio-Rad</td>
			<td>1450021</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Gibco</td>
			<td>15240096</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27</td>
			<td>Gibco</td>
			<td>A3582801</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type 2</td>
			<td>Worthington</td>
			<td>LS004176</td>
			<td>use 1,400 U/mL</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma Aldrich</td>
			<td>67685</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute &#8805;99.5%</td>
			<td>VWR</td>
			<td>VWRC83813.360</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (low endotoxin)</td>
			<td>Biowest</td>
			<td>S1810-500</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030024</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin 10%</td>
			<td>Sigma-Aldrich</td>
			<td>A1595-50ML</td>
			<td>Cell wash buffer</td>
		</tr>
		<tr>
			<td>DPBS (Ca2+, Mg2+free)</td>
			<td>Gibco</td>
			<td>14190-169</td>
			<td>Cell wash buffer</td>
		</tr>
		<tr>
			<td>Magnesium Chloride Solution, 1 M</td>
			<td>Sigma-Aldrich</td>
			<td>M1028</td>
			<td>Nucleus Lysis buffer</td>
		</tr>
		<tr>
			<td>Nonidet P40 Substitute (nonionic detergent)</td>
			<td>Sigma-Aldrich</td>
			<td>74385</td>
			<td>Nucleus Lysis buffer</td>
		</tr>
		<tr>
			<td>Nuclease free water (not DEPC-treated)</td>
			<td>Invitrogen</td>
			<td>AM9937</td>
			<td>Nucleus Lysis buffer</td>
		</tr>
		<tr>
			<td>Protector RNase Inhibitor, 40 U/&#181;L</td>
			<td>Sigma-Aldrich</td>
			<td>3335399001</td>
			<td>Nucleus Lysis buffer</td>
		</tr>
		<tr>
			<td>Sodium Chloride Solution, 5 M</td>
			<td>Sigma-Aldrich</td>
			<td>59222C</td>
			<td>Nucleus Lysis buffer</td>
		</tr>
		<tr>
			<td>Trizma Hydrochloride Solution, 1 M, pH 7.4</td>
			<td>Sigma-Aldrich</td>
			<td>T2194</td>
			<td>Nucleus Lysis buffer</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin 10%</td>
			<td>Sigma-Aldrich</td>
			<td>A1595-50ML</td>
			<td>Nucleus wash</td>
		</tr>
		<tr>
			<td>DPBS (Ca2+, Mg2+free)</td>
			<td>Gibco</td>
			<td>14190-169</td>
			<td>Nucleus wash</td>
		</tr>
		<tr>
			<td>Protector RNase Inhibitor,40 U/&#181;L</td>
			<td>Sigma-Aldrich</td>
			<td>3335399001</td>
			<td>Nucleus wash</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin 10%</td>
			<td>Sigma-Aldrich</td>
			<td>A1595-50ML</td>
			<td>ST staining buffer (ST-SB)</td>
		</tr>
		<tr>
			<td>Calcium chloride solution, 1 M</td>
			<td>Sigma-Aldrich</td>
			<td>21115-100ML</td>
			<td>ST staining buffer (ST-SB)</td>
		</tr>
		<tr>
			<td>Magnesium Chloride Solution, 1 M</td>
			<td>Sigma-Aldrich</td>
			<td>M1028</td>
			<td>ST staining buffer (ST-SB)</td>
		</tr>
		<tr>
			<td>Nuclease free water (not DEPC treated)</td>
			<td>Invitrogen</td>
			<td>AM9937</td>
			<td>ST staining buffer (ST-SB)</td>
		</tr>
		<tr>
			<td>Sodium Chloride Solution, 5M</td>
			<td>Sigma-Aldrich</td>
			<td>59222C</td>
			<td>ST staining buffer (ST-SB)</td>
		</tr>
		<tr>
			<td>Trizma Hydrochloride Solution, 1M, pH 7.4</td>
			<td>Sigma-Aldrich</td>
			<td>T2194</td>
			<td>ST staining buffer (ST-SB)</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Merck Millipore</td>
			<td>822184</td>
			<td>ST staining buffer (ST-SB)</td>
		</tr>
		<tr>
			<td>TotalSeq-A0451 anti-Nuclear Pore Complex Proteins Hashtag 1 Antibody</td>
			<td>Biolegend</td>
			<td>682205</td>
			<td>Hashtag antibody</td>
		</tr>
		<tr>
			<td>TotalSeq-A0452 anti-Nuclear Pore Complex Proteins Hashtag 2 Antibody</td>
			<td>Biolegend</td>
			<td>682207</td>
			<td>Hashtag antibody</td>
		</tr>
		<tr>
			<td>TotalSeq-A0453 anti-Nuclear Pore Complex Proteins Hashtag 3 Antibody</td>
			<td>Biolegend</td>
			<td>682209</td>
			<td>Hashtag antibody</td>
		</tr>
		<tr>
			<td>TotalSeq-A0461 anti-Nuclear Pore Complex Proteins Hashtag 11 Antibody</td>
			<td>Biolegend</td>
			<td>682225</td>
			<td>Hashtag antibody</td>
		</tr>
		<tr>
			<td>TotalSeq-A0462 anti-Nuclear Pore Complex Proteins Hashtag 12 Antibody</td>
			<td>Biolegend</td>
			<td>682227</td>
			<td>Hashtag antibody</td>
		</tr>
		<tr>
			<td>TotalSeq-A0463 anti-Nuclear Pore Complex Proteins Hashtag 13 Antibody</td>
			<td>Biolegend</td>
			<td>682229</td>
			<td>Hashtag antibody</td>
		</tr>
		<tr>
			<td>TotalSeq-A0464 anti-Nuclear Pore Complex Proteins Hashtag 14 Antibody</td>
			<td>Biolegend</td>
			<td>682231</td>
			<td>Hashtag antibody</td>
		</tr>
		<tr>
			<td>TotalSeq-A0465 anti-Nuclear Pore Complex Proteins Hashtag 15 Antibody</td>
			<td>Biolegend</td>
			<td>682233</td>
			<td>Hashtag antibody</td>
		</tr>
		<tr>
			<td>TruStain FcX (human)</td>
			<td>Biolegend</td>
			<td>422302</td>
			<td>FC receptor blocking solution</td>
		</tr>
		<tr>
			<td>Equipment and consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bright-Line&#160;Hemacytometer</td>
			<td>Merck</td>
			<td>Z359629-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5702/R A-4-38</td>
			<td>Eppendorf</td>
			<td>&#160;EP022629905</td>
			<td></td>
		</tr>
		<tr>
			<td>CoolCell LX Cell Freezing Container</td>
			<td>Corning</td>
			<td>CLS432003-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryovial</td>
			<td>Thermo Scientific</td>
			<td>479-6840</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA LoBind 0.5 mL Eppendorf tube</td>
			<td>Eppendorf</td>
			<td>EP0030108035-250EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes 1.5 mL</td>
			<td>Eppendorf</td>
			<td>30121872</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 35 mm Not TC-treated Petri dish</td>
			<td>Corning</td>
			<td>351008</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 15 mL Conical Centrifuge Tubes</td>
			<td>Fisher scientific</td>
			<td>10773501</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps Dumont #5</td>
			<td>Fine science tools</td>
			<td>11252-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Hardened Fine Scissors</td>
			<td>Fine science tools</td>
			<td>14091-09</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Ice Pan, rectangular 4 L Orange</td>
			<td>Corning</td>
			<td>CLS432106-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica MS5</td>
			<td>Leica</td>
			<td></td>
			<td>Microscope</td>
		</tr>
		<tr>
			<td>Moria MC50 Scissors</td>
			<td>Fine science tools</td>
			<td>15370-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Noyes Spring Scissors</td>
			<td>Fine science tools</td>
			<td>15012-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus CK2 ULWCD</td>
			<td>Olympus</td>
			<td></td>
			<td>Microscope</td>
		</tr>
		<tr>
			<td>P10</td>
			<td>Gilson</td>
			<td>F144802</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000</td>
			<td>Gilson</td>
			<td>F123602</td>
			<td></td>
		</tr>
		<tr>
			<td>P200</td>
			<td>Gilson</td>
			<td>F123601</td>
			<td></td>
		</tr>
		<tr>
			<td>Preseparation Filters (30 &#181;m)</td>
			<td>Miltenyi biotec</td>
			<td>Miltenyi biotec130-041-407</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking water bath</td>
			<td>GFL</td>
			<td>1083</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon plate</td>
			<td>RubberBV</td>
			<td>3530</td>
			<td>Dissection board</td>
		</tr>
		<tr>
			<td>Software and packages</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell ranger</td>
			<td>V4.0.0</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>R programming</td>
			<td>V4.1.1</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>R sudio</td>
			<td>V1.3.1073</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Seurat</td>
			<td>V4.0</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>tydiverse</td>
			<td>V1.3.1</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Animals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B6.Cg-Tg(Wnt1-cre)2Sor/J mouse</td>
			<td>The Jackson Laboratory</td>
			<td>JAX stock #022501</td>
			<td></td>
		</tr>
		<tr>
			<td>B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J mouse</td>
			<td>The Jackson Laboratory</td>
			<td>JAX stock #007576</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J mice</td>
			<td>Charles River</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Code for the data analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>https://github.com/rubenmethorst/Single-cell-SCG_JoVE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Quality control analysis of the single-nucleus cDNA library preparation and snRNA-seq 
Representative results describe sequencing results of 10,000 captured nuclei in a single pool with a 25,000 reads/nucleus gene expression library and a 5,000 reads/nucleus hashtag library. Figure 3B illustrates the quality control results of the 1st strand cDNA, gene expression (GEX) library, and HTO library, which were checked with Bioanalyzer. The HTO-derived cDNAs are ex...

Watch this Scientific Journal Video about Low-input Nucleus Isolation and Multiplexing with Barcoded Antibodies of Mouse Sympathetic Ganglia for Single-nucleus RNA Sequencing at JoVE.com

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

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 ...

In this JoVE protocol, we use droplet-based single-nucleus RNA sequencing to characterize the biological architecture of cardiac sympathetic neurons in health and disease. This method enables the sequencing of a large collection of neuronal samples over a period of time. By pulling nuclei barcoded with hashtag oligos, this method allows the multiplexing of the samples.This method holds the potential to be extended to sequencing neurons of the human ganglia, which has been challenging due to its large cell size and difficult simultaneous collection of samples. Demonstrating the procedure will be Conny van Munsteren, research technician, and Lieke van Roon, PhD students from our lab Begin by collecting all the materials needed for dissection, then fix the mouse on the dissection board. Wet the mouse with 70%ethanol to minimize the dispersion of fur.Open the skin of the neck region by making a midline cut with scissors. Under a stereo microscope, move the sub-mandibular glands aside and remove the sternal mastoid muscle to expose the common carotid artery and its bifurcation, as well as the superior cervical ganglia and remove the tissue attached to it. Dissect the right and left carotid artery bifurcation and the tissue attached to it.Transfer each dissected piece of tissue to a separate 3.5-centimeter Petri dish containing cold PBS. Locate the superior cervical ganglia attached to the carotid bifurcation. Clean the superior cervical ganglia by removing the artery and the attached tissue.To dissect the stellate ganglia, make a midline cut in the abdomen, followed by opening the diaphragm and the ventral thoracic wall. Remove the lungs, followed by the heart, to expose the dorsal thorax. At the level of the first rib, locate the left and right stellate ganglia anterolateral to the musculus colli longus.Dissect both stellate ganglia with forceps by removing the surrounding tissue. Transfer them to 3.5-centimeter Petri dishes containing cold PBS. Using forceps, transfer the superior cervical ganglia and stellate ganglia to separate 1.5-milliliter micro-centrifuge tubes then add 500 microliters of 0.25%Trypsin-EDTA solution to each tube and incubate in a shaking water bath.Allow the ganglia to settle at the bottom of the micro-centrifuge tubes. Transfer the supernatant to a 15-milliliter tube containing five milliliters of ganglion medium. To the micro-centrifuge tubes, add 500 microliters of collagenase type 2 solution and incubate in a shaking water bath.After incubation, resuspend the ganglia in collagenase solution by pipetting until tissue clumps are no longer detected. Transfer the cell suspension to the previously used 15-milliliter tube containing the ganglia culture medium with Trypsin-EDTA suspension. Centrifuge the cell suspension and discard the cell supernatant.Resuspend the pellet in 270 microliters of fetal bovine serum and transfer it to a 1-milliliter cryovial. To count the cells, mix 5 microliters of the ganglionic cell suspension with five microliters of 0.4%trypan blue dye. Load the mixture into a hemocytometer and count the total and live cell numbers under a microscope.Next, add 30 microliters of dimethyl sulfoxide to each cryovial and mix well. Transfer the cryovials into a cell-freezing container and place it at minus 80 degrees Celsius overnight. Prepare 15-milliliter tubes with a 30-micrometer strainer on top.Pre-rinse the strainer with 1 milliliter of ganglion medium. Take out the cryovials from liquid nitrogen and immediately thaw them in a water bath at 37 degrees Celsius till a small pellet of ice is left in the cryovial. Add 1 milliliter of the ganglion medium into each cryovial while carefully shaking the vial to recover the ganglionic cells.Load the ganglionic cell suspension on the strainer and rinse with 4 to 5 milliliters of ganglion medium. Centrifuge the strained cell suspension. Remove the supernatant and resuspend the cells in 50 microliters of cell wash buffer.Transfer the cell suspension to a 0.5-milliliter low-binding micro-centrifuge tube. Centrifuge the cell suspension. A centrifuge with a swinging bucket rotor is essential from now on.After centrifugation, remove 45 microliters of the supernatant without dislodging the cell pellet. Add 45 microliters of chilled lysis buffer, gently mixed by pipetting, and incubate the cells for 8 minutes on ice, then add 50 microliters of cold nuclei wash buffer to each tube. Centrifuge the nuclei suspension and remove 95 microliters of the supernatant without disrupting the nuclei pellet.Add 45 microliters of chilled nuclei wash buffer to the pellet, repeat the centrifugation, and remove the supernatant. To the nucleus pellet, add 50 microliters of ST-SB and gently pipette until the nuclei are completely resuspended, then add 5 microliters of FC blocking reagent and incubate for 10 minutes on ice. Next, add 1 microliter of single-nucleus hashtag antibody and incubate for 30 minutes on ice.After incubation, add 100 microliters of ST-SB to each tube. Centrifuge the suspension and remove 145 microliters of the supernatant without disrupting the nuclei pellet. Repeat the centrifugation and remove the supernatant.Resuspend the nuclei pellet in 50 microliters of ST-SB and mix gently. Take 5 microliters of the nuclei suspension and mix it with 5 microliters of 0.4%trypan blue to count the nuclei under a microscope. Again, centrifuge the nuclei suspension and resuspend the nuclei pellet in ST-SB to achieve a target nuclei concentration of 1, 000 to 3, 000 nuclei per microliter.Pull the samples to achieve the desired number of cells. Quality control analysis for library preparation showed hashtag oligo-derived cDNAs smaller than 180 base pairs. A broad peak from 300 to 1, 000 base pairs represented a high-quality gene expression library, whereas a specific peak of 194 base pairs showed the hashtag oligo library.Single-nucleus RNA sequencing analysis revealed 12 clusters that were visualized by UMAP and hashtag antibodies were distributed among all clusters. Specific labeling by hashtag oligos was confirmed by the expression of Xist. Hashtags 1 to 4 labeled female, while 5 to 8 labeled male samples.The quality and resolution of the sequencing data were verified by key transcripts such as TH, DBH, and SNAP25 in clusters 5 and 7 for sympathetic neurons, S100B in clusters 0 to 3 for satellite glial cells, PECAM 1 in cluster 4 for endothelial cells, and ACTA2 in cluster 8 for stromal cells. When attempting this method, it is important to remember the proper preparation and adequate equipment such as fine dissection scissors, sharp tweezers and a centrifuge with a swinging bucket rotor are essential. The stepwise approach for single-nucleus RNA sequencing of sympathetic extrinsic cardiac ganglia has the potential for broad application in characterizing the ganglia innervating other related organs and tissues in health and disease.

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