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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 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.
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.
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
2. Tissue dissection and dissociation
3. Gradient centrifugation
4. Isolation of nuclei
5. Single nuclei RNA and ATAC seq
Single nucleus 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...
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...
The authors have nothing to disclose.
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.).
Name | Company | Catalog Number | Comments |
2-Mercaptoethanol | Sigma | M6250 | |
CaCl2 | Sigma | 21115-100ML | |
Dounce Homogenizer | Active motif | 40401 | |
EDTA (0.5 M) | Thermo Scientific | R1021 | |
Falcon 5 mL Round Bottom Polystyrene Test Tube | Corning | 352235 | |
Iodixanol (aka Optiprep) | Stem cell technologies | 07820 | |
MACs Smart Strainers (30 µm) | Miltenyi Biotec | 130-098-458 | |
MACS SmartStrainers (100 µm) | Miltenyi Biotec | 130-098-463 | |
Mg(Ac)2 | Sigma | 63052-100ML | |
NP-40 | Abcam | ab142227 | |
Nuclei Isolation Kit: Nuclei EZ Prep | Sigma | NUC101-1KT | |
Phenylmethanesulfonyl fluoride (PMSF) | Sigma | P7626 | |
Pre-Separation Filters (20 µm) | Miltenyi Biotec | 130-101-812 | |
Safe lock tubes 1.5 mL | Eppendorf | 0030120086 | |
Safe lock tubes 2.0 mL | Eppendorf | 0030120094 | |
Single Cell ATAC | 10x Genomics | ||
Single Cell Gene Expression | 10x Genomics | ||
Sucrose | Sigma | S0389 | |
Wide Bore pipette tips (1000 µL) | Themo Fisher Scientific | 2079GPK | |
Wide Bore pipette tips (200 µL) | Themo Fisher Scientific | 2069GPK |
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