Single Nuclei Isolation from Coronary Endarterectomy Tissue of Coronary Artery Bypass Graft Patients

Our major research focus is to map the cellular diversity in unstable atherosclerotic plaques to understand cell-to-cell interactions, and then develop RNA-based prognostic markers. Membrane fragility hinders single cell isolation from the tissue obtained during endarterectomy. Therefore, this protocol involves isolating of preserved nuclei, enabling transcriptomic profiling of high-risk plaques.

To begin, gather all the reagents and icebox to perform the procedure. Wash the tissue once or twice with serum-free RPMI medium to remove bloodstains and fragments. Using sterile forceps, transfer the tissue onto a sterile Petri dish and record the weight.

Using a surgical blade number 10, mince the tissue into small pieces of approximately one cubic millimeter in size. Now add one milliliter chilled lysis buffer, and the tissue to a 1.5 milliliter micro centrifuge tube. Lice the tissue on ice using a micro pestle by applying a gentle, consistent force and swirling the micro pestle clockwise and anti-clockwise alternately.

Continue lysis for approximately 30 minutes while keeping the sample on ice. Filter the solution through a 100 micrometer pore size strainer into a 15 milliliter centrifuge tube to remove debris and tissue chunks. Centrifuge the nuclei at 500 G for five minutes at four degrees Celsius using a swinging bucket rotor.

Then remove the supernatant using a regular bore pipette tip without disturbing the pellet. Chill the nuclei wash in resuspension buffer on ice before use. Resuspend the pellet in the chilled nuclei wash and resuspension buffer.

Quickly assess lysis efficiency and cell viability by adding five microliters Trypan blue to an aliquot of the sample and counting the cells using a hemocytometer. If viable cells are observed under the microscope, repeat the lysis, filtration, and centrifugation steps. Then filter the solution through a 30 micrometer pore size strainer into a 15 milliliter centrifuge tube to remove debris and clumps.

Centrifuge the nuclei at 500 G for five minutes at four degrees Celsius using a swinging bucket rotor. After centrifugation, remove the supernatant using a regular bore pipette tip without disturbing the pellet and resuspend the pellet in one milliliter nuclei wash and resuspension buffer. Take 15 microliters of the isolated nuclei suspension in a 1.5 milliliter micro centrifuge tube.

Add five microliters Trypan blue dye at 0.4%weight per volume to the nuclei suspension and mix gently by pipetting. After a five minute incubation, load 10 microliters of the stained mixture onto a hemocytometer. Observe the sample under a compound inverted microscope to examine the morphology and count the number of nuclei.

Take one milliliter of the isolated nuclei suspension and centrifuge at 500 G for five minutes at four degrees Celsius using a swinging bucket rotor. Remove the supernatant using a regular bore pipette tip without disturbing the pellet. Resuspend the pellet in 200 microliters fluorescence-activated cell sorting buffer composed of PBS and 1%bovine serum albumin.

Divide the 200 microliters of resuspended nuclei into two aliquots. Keep 100 microliters as the unstained control and use the remaining 100 microliters as the stained sample. Add one microliter DAPI from a one milligram per milliliter stock solution to the stained sample to achieve a final concentration of 10 micrograms per milliliter.

Mix gently by pipetting and incubate at room temperature in the dark for five minutes. Set the flow cytometer to use the violet laser at 405 nanometers and detect using 448 by 45 nanometers. Run the unstained control sample first and acquire a minimum of 10, 000 events of the total population.

Generate a plot of forward scatter area versus forward scatter height to distinguish singlets from doublets and aggregates. Draw a gate around events displaying a linear relationship to retain only singlet nuclei. Next, generate a bivariate plot of forward scatter area versus side scatter area, and apply a broad gate encompassing the entire scatter population.

Now, create a 2D density pseudo color plot of side scatter area versus violet 450 nanometer area. Use the unstained control to define background fluorescence and guide gate placement. Identify and gate the distinct DAPI positive population corresponding to intact nuclei.

Take 50 microliters of nuclei suspension and add one microliter DAPI from a one milligram per milliliter stock solution to achieve a final concentration of 10 micrograms per milliliter. Mix gently by pipetting and incubate at room temperature in the dark for five minutes before confocal imaging. Mount five microliters of the stained nuclei suspension onto a clean glass slide and cover with a cover slip.

Next, open the confocal microscope software and observe the nuclei in 40x bright field. Finally, acquire confocal images using the laser scanning confocal microscope. Excite DAPI using a 405 nanometer laser and collect emission between 429 and 474 nanometers.

Following nuclear isolation and Trypan blue staining, a distinct population of nuclei was observed without intact viable cells, confirming effective tissue lysis. Isolated nuclei appeared as dense, round, dark blue bodies under the microscope, indicating successful nuclear isolation. Calcified plaque tissue produced lower nuclear yield and reduced nuclear quality.

Flow cytometry of the unstained control detected 77.88%singlets and 0.19%DAPI positive events, reflecting baseline autofluorescence. The DAPI stained sample showed 68.1%singlets and 25.31%DAPI positive nuclei, indicating a clear fluorescence shift. Histogram analysis showed a rightward fluorescence shift of the DAPI-stained nuclei relative to the unstained nuclei population.

Confocal imaging of the stained sample showed bright blue fluorescent nuclei that appeared intact and uniformly labeled. Single nuclei RNA sequencing analysis identified 16 distinct cell clusters following dimensionality reduction. The most significant challenge while performing this protocol is high calcification in this tissue, which critically impairs the nuclei quality and yield.

Facial transcriptome is another approach which we can use to get detailed information, so it will give us the information about special gene expression as well as cell-to-cell interaction.