A Simple, Quick, and Partially Automated Protocol for the Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nucleus Sequencing

This research focuses on supporting drug discovery studies using single nuclei RNA sequencing to investigate the efficacy and safety of potential treatments. Therefore, we are developing standardized nuclei extraction protocols for fresh frozen tissue samples from various organs. Our goal was to automate and standardize nuclei extraction to achieve consistent and reliable results by reducing non-biological variability.

The advantage is the partial automation through the use of a robotic dissociator and the simple and short workflow that can be applied on various tissue samples of different species. Having standardized protocols can help us answer questions about drug biodistribution, as well as explore the mechanisms underlying any safety signals that we might observe. In the future, we will be testing the applicability of our protocol for other downstream applications, such as ATAC-seq or multiomics.

We will also try to establish protocols to detect virally expressed transgenes in single nuclei. To begin, start the robotic disassociator. Turn on the cooling by setting the slider at the top of the right of the screen to cool and clicking on it to start cooling so that the slider appears orange.

Next, check that the attached NSR and NIB bottles have sufficient liquid and are properly cooled. Remove the tissue samples from the minus 80 degrees Celsius freezer and immediately place them on dry ice. Using a pre-cooled scalpel, cut the tissue into a 15 to 15 milligram piece on a pre-cooled Petri dish or metal plate placed on dry ice.

Remove the nuclei isolation cartridge from the refrigerator and unpack it. Remove the grinder and pipette 15 microliters of RNAs inhibitor to the bottom of the cartridge. Using tweezers, place the tissue sample at the bottom of the cartridge.

Next select run a protocol on the instrument and click on the nuclei option in the upper left corner. From the menu, select the low volume nuclei isolation protocol and then click on modify to verify that the disruption speed is set to fastest. Then open the instrument door, insert the cartridge into the designated location, rotate the cartridge lock, and slide into the stage until the red knob clicks into place.

Close the door and click next to start the nuclei extraction run. Once the run is finished, remove the cartridge from the instrument by lifting the red knob and pulling out the stage. Immediately place the cartridge on ice.

After isolating single nuclei from frozen mammalian tissues, carefully pierce the round foil on the dissociator cartridge using a pipette tip. To facilitate sucrose gradient cleanup, remove 900 microliters of the nuclei suspension from the cartridge and add it to 500 microliters of sucrose cushion solution, or SCS, prepared in a two milliliter tube. Mix well by pipetting until the mixture is homogenous.

Next, hold the tube at an angle and carefully add 1, 400 microliters of nuclei suspension-SCS mixture dropwise onto a new 500 microliters of SCS, creating a clearly visible phase separation. Carefully close the tube and place it back on the ice without disturbing the phase separation. Carefully spin the tubes at 13, 000 G for 15 minutes at four degrees Celsius in a pre-cooled centrifuge.

Remove the supernatant without disturbing the pellet and carefully resuspend the pellet in 50 microliters of ice cold NSR. Pull the two pellets of the same sample in a new 1.5 milliliter tube. Add 900 microliters of ice cold NSR to a total volume of one milliliter and mix well by pipetting.

Centrifuge the sample at 500 G for five minutes at four degrees Celsius with a swinging bucket rotor centrifuge. Remove the supernatant and resuspend the pellet in 100 microliters of PBS. For counting, dilute 10 microliters of nuclei suspension in 20 microliters of PBS.

Add 25 microliters of propidium iodide staining solution to a mixing well of the fluorescent counter counting plate. Then add 25 microliters of diluted nuclei suspension and mix well by pipetting. Transfer 50 microliters of the stained sample from the mixing well to the loading well.

Load the counting plate on the cell counter and start the count. After counting, dilute the samples with PBS to the desired concentration for single nuclei RNA sequencing. Using the partially automated nuclei isolation protocol, good quality nuclei were obtained without signs of blabbing, debris, or clumping.

Violin plots representing the distribution of gene counts, UMI counts, and mitochondrial content in each sample are shown. Expected cell types from each tissue were identified and all animals contributed to all clusters indicating low technical variability introduced by the protocol. Furthermore, cellular proportions, UMI counts, and gene counts were comparable across all three samples per tissue type.