Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq

The goal of our method is to isolate nuclei from fresh frozen tumors and the same nuclei preparation can be used for transcriptional and epigenetic studies. Therefore, we can ask several key questions such as, how do tumors evolve during progression and how do therapies affect tumor composition? The biggest advantage of this method is that it is simple, reduces processing time, and most importantly, yields high-quality nuclei.

The absence of a sorting step reduces stress on the nuclei. And the other advantage is its ability to be used on archival frozen tumors. Demonstrating the procedure will be Ashwin Narayanan, a postdoctoral fellow from my laboratory.

Begin by transferring 10 to 60 milligrams of fresh frozen tissue sample to a pre-chilled Petri dish. Mince or chop the fresh frozen tissue with a razor blade into small pieces on ice. Add 500 microliters of chilled nuclei lysis buffer onto the tissue in the Petri dish.

Then transfer the mixture to a douncer. Dounce the tissue pieces with the loose pestle for about 20 strokes until friction is reduced. Then dounce it with the tight pestle for 20 strokes to achieve complete tissue homogenization.

Transfer the homogenate into a pre-chilled two milliliter tube and add one milliliter of chilled lysis buffer into the douncer, rinse it and add it to the tube. Mix it gently and incubate it on ice for five minutes, mixing with a wide bore pipette tip one to two times during the incubation. Filter the entire homogenate using a 30 microliter strainer mesh and collected into a 15 milliliter Falcon tube.

Then transfer it back into a new pre-chilled two milliliter tube. A single strainer is typically sufficient for the entire homogenate. Check the sample under a light microscope to verify the removal of large debris and the intactness of the nuclear membrane.

Nuclei need not be round and the nuclear membrane should not be distorted. If debris is present, repeat the filtration. Next, centrifuge the nuclei on a bench top centrifuge for five minutes.

Remove the supernatant, leaving behind approximately 50 microliters of the nuclei-containing pellet. Gently resuspend the pellet in another milliliter of nuclei lysis buffer and incubate it for five minutes on ice. Repeat the centrifugation, then remove the supernatant without disturbing the pellet.

Add 500 milliliters of HB and incubate the sample for five minutes without re-suspending. Then resuspend the nuclei in another one milliliter of HB.Centrifuge for another five minutes, then remove the supernatant. Resuspend the nuclei in 200 microliters of HB and transfer the suspension into a new two milliliter tube.

Add 200 microliters of 50%iodixanol solution to the nuclei for a final concentration of 25%iodixanol and mix 10 times with the pipette set to 300 microliters. Then add 300 microliters of 29%iodixanol solution under the 25%mixture using a P1000 fine tip to avoid mixing the layers. Add 300 microliters of 35%iodixanol solution under the 29%mixture with a P1000 fine tip, then slowly remove it.

Place the samples in a swinging bucket centrifuge and spin them for 20 minutes at 3, 500 G at four degrees Celsius with the break off. Gently remove the samples without shaking and observe them under light. A clear white band of 95%pure nuclei should be visible between the second and third layer.

To isolate the nuclei, aspirate the top layers to expose the white nuclei band at the interface. Collect the nuclei band in a 200 milliliter volume and transfer it to a fresh tube. Then filter it with a 20 micrometer filter.

Check the nuclei under a light microscope to verify the removal of large debris and the intactness of the nuclear membrane. They should be round and the nuclear membrane should not be distorted. Count the nuclei using Trypan Blue staining on a hemocytometer and aliquot them for snRNA-seq or snATAC-seq.

Various filtration steps along with the gradient centrifugation allow for the isolation of pure nuclei with the majority of debris discarded. The same isolated nuclei preparation can be used for both single nuclei RNA-seq and single nuclei ATAC-seq. Since the isolated nuclei are from the same sample, the data generated can be co-embedded using R packages such as the Seurat to generate clusters and provide a multiomics perspective.

To determine whether the protocol is comparable to published datasets, the obtained data was compared with four publicly available single nuclei RNA-seq studies related to the central nervous system. All datasets were merged and the distribution of the number of UMIs and genes were visualized using a violin plot. The result indicated that the method is comparable to the latest snRNA-seq protocol.

When attempting this protocol, it is important to be extra careful and not mix the layers, during gradient centrifugation and nuclei isolation.