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

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.