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.

low-input-nucleus-isolation-multiplexing-with-barcoded-antibodies

Research

JoVE Journal

Medicine

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified version of carotid partial ligation methods [1,2] to show that it acutely induces low and oscillatory flow conditions, two key characteristics of disturbed flow, in the mouse common carotid artery. Using this model, we have provided direct evidence that disturbed flow indeed leads to rapid and robust atherosclerosis development in Apolipoprotein E knockout mouse [3]. We also developed a method of endothelial RNA preparation with high purity from the mouse carotid intima [3]. Using this mouse model and method, we found that partial ligation causes endothelial dysfunction in a week, followed by robust and rapid atheroma formation in two weeks in a hyperlipidemic mouse model along with features of complex lesion formation such as intraplaque neovascularization by four weeks. This rapid in vivo model and the endothelial RNA preparation method could be used to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases. Also, it could be used to test various therapeutic interventions targeting endothelial dysfunction and atherosclerosis in considerably reduced study duration.

This describes a partial carotid ligation surgery, which causes disturbed flow conditions and subsequent atherosclerosis development (in two weeks) with intraplaque neo-vascularization (in four weeks) in the mouse common carotid artery. We also describe a novel method of RNA isolation from the carotid intima, providing high purity endothelial RNA.

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified ...

Sequencing of Bacterial Microflora in Peripheral Blood: our Experience with HIV-infected Patients

The healthy gastrointestinal tract is physiologically colonized by a large variety of commensal microbes that influence the development 
	of the humoral and cellular mucosal immune system1,2. 
 Microbiota is shielded from the immune system via a strong mucosal barrier. Infections and antibiotics are known to alter both the normal 
	gastrointestinal tract barrier and the composition of resident bacteria, which may result in possible immune abnormalities3.
HIV causes a breach in the gastrointestinal barrier with progressive failure of mucosal immunity and leakage into the systemic circulation of bacterial bioproducts, such as lipopolysaccharide and 
	bacterial DNA fragments, which contribute to systemic immune activation4-7. Microbial translocation is implicated in HIV/AIDS immunopathogenesis and response to therapy 4,8.
We aimed to characterise the composition of bacteria translocating in peripheral blood of HIV-infected patients. To pursue our aim we set up a PCR reaction for the panbacteric 16S ribosomial gene 
	followed by a sequencing analysis.
Briefly, whole blood from both HIV-infected and healthy subjects is used. Given that healthy individuals present normal intestinal homeostasis no translocation of microflora is expected in 
	these patients. Following whole blood collection by venipuncture and plasma separation, DNA is extracted from plasma and used to perform a broad range PCR reaction for the panbacteric 
	16S ribosomial gene9. Following PCR product purification, cloning and sequencing analyses are performed.

Our experiment will show how to perform a sequencing analysis of bacterial species translocating in peripheral blood of HIV positive patients.

The healthy gastrointestinal tract is physiologically colonized by a large variety of commensal microbes that influence the development 
	of the humoral ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

Unbiased Deep Sequencing of RNA Viruses from Clinical Samples

Here we outline a next-generation RNA sequencing protocol that enables de novo assemblies and intra-host variant calls of viral genomes collected from clinical and biological sources. The method is unbiased and universal; it uses random primers for cDNA synthesis and requires no prior knowledge of the viral sequence content. Before library construction, selective RNase H-based digestion is used to deplete unwanted RNA &#8212; including poly(rA) carrier and ribosomal RNA &#8212; from the viral RNA sample. Selective depletion improves both the data quality and the number of unique reads in viral RNA sequencing libraries. Moreover, a transposase-based &#39;tagmentation&#39; step is used in the protocol as it reduces overall library construction time. The protocol has enabled rapid deep sequencing of over 600 Lassa and Ebola virus samples-including collections from both blood and tissue isolates-and is broadly applicable to other microbial genomics studies.

This protocol describes a rapid and broadly applicable method for unbiased RNA-sequencing of viral samples from human clinical isolates.

Here we outline a next-generation RNA sequencing protocol that enables de novo assemblies and intra-host variant calls of viral genomes collected from ...

Novel Approach for Simultaneous Recording of Renal Sympathetic Nerve Activity and Blood Pressure with Intravenous Infusion in Conscious, Unrestrained Mice.

Renal sympathetic nerves contribute significantly to both physiological and pathophysiological phenomena. Evaluating renal sympathetic nerve activity (RSNA) is of great interest in many areas of research such as chronic kidney disease, hypertension, heart failure, diabetes and obesity. Unequivocal assessment of the role of the sympathetic nervous system is thus imperative for proper interpretation of experimental results and understanding of disease processes. RSNA has been traditionally measured in anesthetized rodents, including mice. However, mice usually exhibit very low systemic blood pressure and hemodynamic instability for several hours during anesthesia and surgery. Meaningful interpretation of RSNA is confounded by this non-physiological state, given the intimate relationship between sympathetic nervous tone and cardiovascular status. To address this limitation of traditional approaches, we developed a new method for measuring RSNA in conscious, freely-moving mice. Mice were chronically instrumented with radio-telemeters for continuous monitoring of blood pressure as well as a jugular venous infusion catheter and custom-designed bipolar electrode for direct recording of RSNA. Following a 48-72 hour recovery period, survival rate was 100% and all mice behaved normally. At this time-point, RSNA was successfully recorded in 80% of mice, with viable signals acquired up to 4 and 5 days post-surgery in 70% and 50% of mice, respectively. Physiological blood pressures were recorded in all mice (116&plusmn;2 mmHg; n=10). Recorded RSNA increased with eating and grooming, as well-established in the literature. Furthermore, RSNA was validated by ganglionic blockade and modulation of blood pressure with pharmacological agents. Herein, an effective and manageable method for clear recording of RSNA in conscious, freely-moving mice is described.

Anesthetized mice exhibit non-physiological systemic blood pressure, which precludes meaningful assessment of autonomic tone given the intimate relationship between blood pressure and the autonomic nervous system. Thus, a novel method to simultaneously record renal sympathetic nerve activity and blood pressure with intravenous infusion in conscious mice is outlined.

Renal sympathetic nerves contribute significantly to both physiological and pathophysiological phenomena. Evaluating renal sympathetic nerve activity ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

Whole Genome Sequencing of Candida glabrata for Detection of Markers of Antifungal Drug Resistance

Candida glabrata can rapidly acquire mutations that result in drug resistance, especially to azoles and echinocandins. Identification of genetic mutations is essential, as resistance detected in vitro can often be correlated with clinical failure. We examined the feasibility of using whole genome sequencing (WGS) for genome-wide analysis of antifungal drug resistance in C. glabrata. The aim was torecognize enablers and barriers in the implementation WGS and measure its effectiveness. This paper outlines the key quality control checkpoints and essential components of WGS methodology to investigate genetic markers associated with reduced susceptibility to antifungal agents. It also estimates the accuracy of data analysis and turn-around-time of testing.
Phenotypic susceptibility of 12 clinical, and one ATCC strain of C. glabrata was determined through antifungal susceptibility testing. These included three isolate pairs, from three patients, that developed rise in drug minimum inhibitory concentrations. In two pairs, the second isolate of each pair developed resistance to echinocandins. The second isolate of the third pair developed resistance to 5-flucytosine. The remaining comprised of susceptible and azole resistant isolates. Single nucleotide polymorphisms (SNPs) in genes linked to echinocandin, azole and 5-flucytosine resistance were confirmed in resistant isolates through WGS using the next generation sequencing.&#160;Non-synonymous SNPs in antifungal resistance genes such as FKS1, FKS2, CgPDR1, CgCDR1 and FCY2 were identified. Overall, an average of 98% of the WGS reads of C. glabrata isolates mapped to the reference genome with about 75-fold read depth coverage. The turnaround time and cost were comparable to Sanger sequencing.
In conclusion, WGS of C. glabrata was feasible in revealing clinically significant gene mutations involved in resistance to different antifungal drug classes without the need for multiple PCR/DNA sequencing reactions. This represents a positive step towards establishing WGS capability in the clinical laboratory for simultaneous detection of antifungal resistance conferring substitutions.

Whole Genome Sequencing of Candida glabrata for Detection of Markers of Antifungal Drug Resistance

This study implemented whole genome sequencing for analysis of mutations in genes conferring antifungal drug resistance in Candida glabrata. C. glabrata isolates resistant to echinocandins, azoles and 5-flucytosine, were sequenced to illustrate the methodology. Susceptibility profiles of the isolates correlated with presence or absence of specific mutation patterns in genes.

Candida glabrata can rapidly acquire mutations that result in drug resistance, especially to azoles and echinocandins. Identification of genetic mutations ...

Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of metabolic syndrome (MetS)-related heart failure with preserved ejection fraction (HFpEF). The prevalence of MetS-related HFpEF is rising, and atrial cardiomyopathies associated with atrial remodeling and atrial fibrillation are clinically highly relevant as atrial remodeling is an independent predictor of mortality. Studies with isolated single-cell cardiomyocytes are frequently used to corroborate and complement in vivo findings. Circulatory vessel rarefication and interstitial tissue fibrosis pose a potentially limiting factor for the successful single-cell isolation of ACMs from animal models of this disease.
We have addressed this issue by employing a device capable of manually regulating the intraluminal pressure of cardiac cavities during the isolation procedure, substantially increasing the yield of morphologically and functionally intact ACMs. The acquired cells can be used in a variety of different experiments, such as cell culture and functional Calcium imaging (i.e., excitation-contraction-coupling).
We provide the researcher with a step-by-step protocol, a list of optimized solutions, thorough instructions to prepare the necessary equipment, and a comprehensive troubleshooting guide. While the initial implementation of the procedure might be rather difficult, a successful adaptation will allow the reader to perform state-of-the-art ACM isolations in a rat model of MetS-related HFpEF for a broad spectrum of experiments.

Here, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes from a rat model of metabolic syndrome-related heart failure with preserved ejection fraction. A manual regulation of intraluminal pressure of cardiac cavities is implemented to yield functionally intact myocytes suitable for excitation-contraction-coupling studies.

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...