We thank the Single Cell Open Lab (scOpenLab) at the German Cancer Center (DKFZ) for helpful discussions. This research was supported by the German Cancer Aid, Max-Eder Program grant number 70111964 (S.T.).

Fresh frozen glioma samples were obtained from the National Center for Tumor diseases (NCT)-tissue bank in Heidelberg, Germany. The use of patient material was approved by the Institutional Review Board at the Medical Faculty of Heidelberg, and informed consent was obtained from all patients included in the study.
1. Experimental preparation
<ol>
	<li>Perform all steps on ice or at 4 &#176;C.</li>
	<li>Pre-chill tubes, dishes, razor blades, Douncers and pestles to 4 &#176;C.</li>
	<li>Prepar...

<ol>
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The field of intra-tumoral heterogeneity is at an exciting stage, with novel assays and platforms being developed to challenge and expand the existing knowledge. Intra-tumoral heterogeneity is a crucial factor that contributes to disease progression and resistance to current treatment modalities in gliomas28. Recent studies on brain tumors have focused on this important aspect by using single cell transcriptomic and epigenomic assays to better characterize the cellular heterogeneity within the sam...

Diffuse lower grade gliomas (LGG), the most common primary brain tumor in adults, are infiltrative neoplasms that often arise in the cerebral hemisphere. LGGs exhibit both inter- and intra-tumor heterogeneity, which is not only driven by the tumor population but also by the non-malignant cells intricately involved in tumor development and progression1,2,3,4,5.
Over the last decade, there has been an avalanche of genomic data gathered in the field of gliomas. These data mainly come from bulk tumor sequencing studies and have contributed immensely to the molecular characterization, and the current classification of brain tumors5,6,7,8,9,10,11. However, even though these studies revealed the broad molecular landscape associated with gliomas, there is still a disappointing lack of progress regarding therapeutic intervention. One of the obstacles to treatment resistance in brain tumors is intra-tumor heterogeneity. To address this issue, various studies have been focusing on the genomic, transcriptomic, proteomic, and epigenetic heterogeneity present within a tumor at a single cell level12,13,14,15,16,17.
Although there have been remarkable technological advancements in the single cell field over the last couple of years, one of the major limiting factors is the availability of fresh specimens needed to isolate the cells and perform these experiments. To overcome this limitation, there have been several successful attempts at performing assays, such as snRNA-seq and snATAC-seq from frozen tissues, using nuclei rather than cells18,19. The majority of these methods rely on either fluorescence-activated cell sorting (FACS) or filtration strategies. Both single cell and single nuclei approaches have their strengths and drawbacks. Single cell approaches maintain mitochondrial transcripts, which although may be informative, can also reduce transcriptome coverage due to their high abundance. Single nuclei isolation approaches eliminate a high percentage of the mitochondrial fraction, thereby allowing a more in-depth coverage of the nuclear transcripts20.
There are various commercially available platforms that have been used over the recent years to assay single cell genomics data, including RNA-seq and ATAC-seq. One of the most prominent platforms is the 10x Genomics Chromium platform for single cell gene expression and single cell ATAC profiling. As the platform works with the help of microfluidic chambers, any debris or aggregates can clog the system leading to loss of data, reagents, and valuable clinical samples. Therefore, the success of single cell studies depends largely on the accurate isolation of single cells/nuclei.
The protocol that we will demonstrate herein is a slightly modified combination of the DroNc-seq and Omni-ATAC-seq protocols, and utilizes a similar approach to recent studies that utilize snRNA-seq to understand neurological disorders and neuronal cell types in the human brain18,19,21,22,23,24. The protocol uses a combination of enzymatic/mechanical dissociation of frozen samples followed by filtration and gradient centrifugation and allows for fast and accurate isolation of single nuclei from fresh frozen glioma tissues. We have successfully used this protocol to generate snRNA-seq and snATAC-seq data from the same nuclei preparation from brain tumor specimens.

<table>
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>21115-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dounce Homogenizer</td>
			<td>Active motif</td>
			<td>40401</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA (0.5 M)</td>
			<td>Thermo Scientific</td>
			<td>R1021</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 5 mL Round Bottom Polystyrene Test Tube</td>
			<td>Corning</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodixanol (aka Optiprep)</td>
			<td>Stem cell technologies</td>
			<td>07820</td>
			<td></td>
		</tr>
		<tr>
			<td>MACs Smart Strainers (30 &#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-098-458</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS SmartStrainers (100 &#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-098-463</td>
			<td></td>
		</tr>
		<tr>
			<td>Mg(Ac)2</td>
			<td>Sigma</td>
			<td>63052-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Abcam</td>
			<td>ab142227</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclei Isolation Kit: Nuclei EZ Prep</td>
			<td>Sigma</td>
			<td>NUC101-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride (PMSF)</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-Separation Filters (20 &#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-101-812</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe lock tubes 1.5 mL</td>
			<td>Eppendorf</td>
			<td>0030120086</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe lock tubes 2.0 mL</td>
			<td>Eppendorf</td>
			<td>0030120094</td>
			<td></td>
		</tr>
		<tr>
			<td>Single Cell ATAC</td>
			<td>10x Genomics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single Cell Gene Expression</td>
			<td>10x Genomics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide Bore pipette tips (1000 &#181;L)</td>
			<td>Themo Fisher Scientific</td>
			<td>2079GPK</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide Bore pipette tips (200 &#181;L)</td>
			<td>Themo Fisher Scientific</td>
			<td>2069GPK</td>
			<td></td>
		</tr>
	</tbody>
</table>

nuclei isolation from fresh frozen brain tumors for single-nucleus rna-seq and atac-seq

Adult diffuse gliomas exhibit inter- and intra-tumor heterogeneity. Until recently, the majority of large-scale molecular profiling efforts have focused on bulk approaches that led to the molecular classification of brain tumors. Over the last five years, single cell sequencing approaches have highlighted several important features of gliomas. The majority of these studies have utilized fresh brain tumor specimens to isolate single cells using flow cytometry or antibody-based separation methods. Moving forward, the use of fresh-frozen tissue samples from biobanks will provide greater flexibility to single cell applications. Furthermore, as the single-cell field advances, the next challenge will be to generate multi-omics data from either a single cell or the same sample preparation to better unravel tumor complexity. Therefore, simple and flexible protocols that allow data generation for various methods such as single-nucleus RNA sequencing (snRNA-seq) and single nucleus Assay for Transposase-Accessible Chromatin with high-throughput sequencing (snATAC-seq) will be important for the field.
Recent advances in the single cell field coupled with accessible microfluidic instruments such as the 10x genomics platform have facilitated single cell applications in research laboratories. To study brain tumor heterogeneity, we developed an enhanced protocol for the isolation of single nuclei from fresh frozen gliomas. This protocol merges existing single cell protocols and combines a homogenization step followed by filtration and buffer mediated gradient centrifugation. The resulting samples are pure single nuclei suspensions that can be used to generate single nucleus gene expression and chromatin accessibility data from the same nuclei preparation.

Single nucleus&#160;genomics is an evolving field with limited data and protocols. A critical factor that influences the outcome of single nuclei assays is the isolation of pure and intact nuclei. We combined two published protocols (DroNc-seq and Omni-ATAC-seq protocols) to isolate high-quality and pure nuclei from fresh frozen glioma tissue blocks in a relatively short time thereby maintaining the stability of the transcripts (Figure 1).
The use of various filtr...

Watch this Scientific Journal Video about Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq at JoVE.com

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

Intra-tumoral heterogeneity is an inherent feature of tumors, including gliomas. We developed a simple and efficient protocol that utilizes a combination of buffers and gradient centrifugation to isolate single nuclei from fresh frozen glioma tissues for single nucleus RNA and ATAC sequencing studies.

Adult diffuse gliomas exhibit inter- and intra-tumor heterogeneity. Until recently, the majority of large-scale molecular profiling efforts have focused ...

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.

nuclei-isolation-from-fresh-frozen-brain-tumors-for-single-nucleus

Research

JoVE Journal

Cancer Research

11.7K 조회수.  University Hospital Heidelberg. 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. ...