This is the first optimized protocol for isolating high-quality nuclei for single-nuclei sequencing experiments using frozen brains of African turquoise killifish, an emerging vertebrate model for aging research. This protocol isolates nuclei very gently which distinguishes it from harsher protocols optimized for other organisms with more myelinated brains, which result in degraded nuclei when used in the killifish. This method will be instrumental in helping us understand brain aging at a single-cell level.
It should translate well to other tissues requiring more gentle nuclei isolation. This protocol is user-friendly. The most demanding task is the gradient centrifugation for debris removal, in which it is better to be slow and exact than fast and messy.
Demonstrating the procedure will be Ari Adler and Brian Teefy, a research lab technician and a post-doctoral scholar from the Benayoun laboratory. After dissecting the killifish, thaw the frozen brain tissue on ice for 10 minutes and dounce the brain in one milliliter of ice-cold nuclei lysis buffer in a two-milliliter dounce homogenizer. While lysing the sample, keep the homogenizer on ice and avoid generating bubbles.
Dounce the tissue as described in the manuscript and let the sample rest on ice in the dounce homogenizer for two minutes. Then, strain the sample through a 70-micrometer cell filter into a 15-milliliter conical tube precoated with 5%BSA. Add four milliliters of nuclei wash buffer to the sample by pipetting the wash buffer over the 70-micrometer cell filter and mix by gently inverting the tube five times.
Using a swinging bucket rotor, centrifuge the sample at 500 G for 10 minutes at four degrees Celsius. Discard the supernatant by gently pouring it into a liquid waste receptacle. Keep the sample upright and remove the remaining wash buffer using a P1000 pipette.
Immediately resuspend the nuclei in one milliliter of nuclei wash buffer with a wide bore P1000 pipette tip. Then, bring the sample to five milliliters by adding four milliliters of nuclei wash buffer and mix as demonstrated previously. Pellet the cell by centrifugation.
Discard the supernatant and resuspend the nuclei in one milliliter of nuclei wash buffer with a wide bore pipette tip. Now, transfer the nuclei to a flow cytometry tube. Add 300 microliters of debris removal solution to the cell suspension and mix it thoroughly by pipetting with a wide bore tip until the mixture is homogeneous.
Hold the tube at a slight angle, and gently overlay one milliliter of nuclei wash buffer on top of the nuclei solution using a P1000 pipette. Centrifuge the sample in a swinging bucket rotor at 3000 G for 10 minutes at four degrees Celsius. Completely discard the top two phases using a P1000 pipette.
Transfer the bottom layer to a 15-milliliter conical tube precoated with 5%BSA. Then, bring the volume to 15 milliliters with nuclei wash buffer and mix by inverting the tube thrice. Centrifuge the tube at 1000G for 10 minutes at four degrees Celsius in a swinging bucket rotor.
Remove the supernatant carefully and immediately resuspend the pellet in 150 microliters of nuclei wash buffer using a wide bore pipette tip. Switch to a standard bore pipette tip set to 300 microliters. Pipette the entire sample and then, attach a 40-micrometer on-tip filter to the end of the pipette tip.
Filter the sample by forcefully expelling the sample through the filter into a low DNA binding, 1.5-milliliter microcentrifuge tube. In a FACS tube, resuspend 10 microliters of the filtered nuclei sample in 90 microliters of the recommended flow cytometry buffer. Stain the samples with propidium iodide at a final concentration of one microgram per milliliter.
After setting up the workspace as described in the manuscript, start the flow by pressing the play icon on the bottom right corner. Check for air bubbles or clumps in the sample using the side scatter area versus HDRT. Also, check the side scatter versus forward scatter plot to verify that the events are accumulating within the window's bounds.
For an enlarged view, double-click the side scatter area versus PerCP-Vio700-A plot. Click the button on the upper left toolbar with a perpendicular line to select a quadrant gate. Then, click anywhere in the side scatter area versus PerCP-Vio700-A plot, and quadrants will appear inside the plot.
Click anywhere on the quadrant dividing lines and slide the cursor to modify the quadrant placement. Place the quadrants such that the upper left quadrant contains debris and the upper right quadrant contains nuclei. Click on the square, gray, I icon to open the properties window.
Go to the region functions tab and ensure a green check mark next to the top item, indicating that the entire plot is selected. Under the functions list, click on count per microliter to produce a green check mark. Click OK, and each quadrant will display a count per microliter metric.
Select count and percent total from the region functions tab to view raw counts and percentages if desired. Draw a polygonal gate around this population by clicking the polygon button on the upper left toolbar. Then, click a single time and slide the mouse to draw a linear segment of the gate.
Click again to finish and begin the next one, followed by a double-click to finish the gate. Click the border of the gate and slide the mouse to reposition the gate. Click the vertices of the gate to modify the shape, and counts per microliter of the gated population will appear.
Healthy nuclei that appeared as singlet nuclei with intact membranes are suitable for downstream analysis. However, unhealthy nuclei are characterized by damaged nuclear membranes, which leads to nuclei clumping and can contribute to background signals in downstream applications. Nuclei are in singlet and multiplet forms in the upper right quadrant, with singlets as the lowest cloud of events followed by doublets and triplets in ascending order.
Debris is marked by events in the two leftmost quadrants in a low-quality nuclei preparation where the debris removal step was omitted. Debris makes up more than 40%of events. However, in a high-quality experiment, debris makes up less than 15%of all the events.
It is imperative to completely remove the middle layer following the debris-removal centrifugation step to reduce contaminating background RNA Nuclei isolated using this protocol can be used for single-nuclei RNA sequencing or ATAC sequencing to obtain single-cell level transcriptomic and epigenetic information. This technique will allow researchers to explore brain gene regulation at the single-cell level in the African turquoise killifish, a naturally short-lived vertebrate model organism for aging research.
