Name: Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq (Video) | JoVE Journal
Uploaded: 2020-08-25T13:45:00.000+00:00
Duration: 6 min 22 s
Description: Learn nuclei isolation from fresh frozen brain tumors for single-nucleus RNA-seq and ATAC-seq using gradient centrifugation. This efficient method yields high-quality nuclei for studying tumor heterogeneity and evolution.

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5 minutes

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>Prepare all buffers in advance. These buffers are stable at room temperature. Sterile filtration is recommended, especially for sucrose. The stock preparations are modified from Corces et al.19. See Tables 1-7.</li>
	<li>Remove samples from liquid nitrogen or -80 &#176;C freezer storage and keep on dry ice until Step 2.1.</li>
</ol>
2. Tissue dissection and dissociation
<ol>
	<li>Transfer fresh frozen tissue sample (10-60 mg) to a pre-chilled Petri dish. Mince/chop fresh frozen tissue with a razor blade to small pieces on ice.</li>
	<li>Add 500 &#181;L of chilled nuclei lysis buffer to a pre-chilled 1.5 mL tube. Place the tissue pieces in the 1.5 mL tube containing the nuclei lysis buffer, and transfer to a Douncer (Table of Materials). 
	NOTE: There are two Dounce homogenizer pestles: &#8220;loose&#8221; or &#8220;A&#8221; pestle for initial sample reduction and &#8220;tight&#8221; or &#8220;B&#8221; pestle for complete sample homogenization.</li>
	<li>Dounce the tissue pieces with the &#8220;loose&#8221; pestle for about 20 strokes, until friction is reduced. If debris is present, the sample may be pre-cleared by filtration with a 100 mm strainer.</li>
	<li>Dounce with the &#8220;tight&#8221; pestle for 20 strokes to achieve complete tissue homogenization.</li>
	<li>Transfer the homogenate (about 500 &#181;L) into a pre-chilled 2 mL tube. Add 1 mL of chilled lysis buffer. Mix gently and incubate on ice for 5 min. Gently mix with a wide-bore pipette tip and repeat 1-2 times during the incubation.</li>
	<li>Filter the entire homogenate using a 30 &#181;m strainer mesh, collect into a 15 mL Falcon&#160;tube and transfer back into a new pre-chilled 2 mL tube. A single strainer is typically sufficient for the entire homogenate.</li>
	<li>Check under a light microscope to verify the removal of large debris and the intactness of the nuclear membrane. Nuclei need to be round and the nuclear membrane should not be distorted. If debris is present, nuclei can be re-filtered.</li>
	<li>Centrifuge the nuclei at 500 x g for 5 min at 4 &#176;C on a bench top centrifuge. Remove the supernatant, leaving behind ~50 &#181;L with pellet containing the nuclei. Gently resuspend the pellet in another 1 mL of nuclei lysis buffer and incubate for 5 min on ice.</li>
	<li>Centrifuge the nuclei at 500 x g for 5 min at 4&#176;C. Remove the supernatant without disturbing pellet, add 500 mL of 1x homogenization buffer (HB) (Table 4) and incubate for 5 min without resuspending. Then, resuspend the nuclei in another 1.0 mL of 1x HB.</li>
	<li>Centrifuge the nuclei at 500 x g for 5 min at 4 &#176;C. Remove the supernatant and gently resuspend the nuclei in 200 &#181;L of 1x HB into a new 2.0 mL tube.</li>
</ol>
3. Gradient centrifugation
<ol>
	<li>Add 200 &#181;L of 50% iodixanol solution (Table 5) to give a final concentration of 25% iodixanol. Mix well 10 times with pipette set on 300 mL.</li>
	<li>Add 300 &#181;L of 29% iodixanol solution (Table 6) under the 25% mixture. Use a P1000 fine tip to avoid mixture of the layers.</li>
	<li>Add 300 &#181;l of 35% Iodixanol solution (Table 7) under the 29% mixture. Use a P1000 fine tip to avoid mixture of the layers. 
	Caution: This step requires gradual removal of the pipette tip during pipetting to avoid excessive volume displacement.</li>
	<li>Place the samples in a swinging bucket centrifuge, spin for 20 min at 3,500 x g at 4&#176;C with the brake off.</li>
	<li>Gently remove the samples without shaking and observe under light. A clear white band of 95% pure nuclei should be visible between the second and third layer (Figure 1).</li>
</ol>
4. Isolation of nuclei
<ol>
	<li>Aspirate the top layers until the white nuclei band at the interphase of 29%-35%.</li>
	<li>Collect the nuclei band in a 200 mL volume, transfer to a fresh tube and filter with a 20 &#181;m filter (Table of Materials). 
	NOTE: The nuclei do not need to be resuspended prior to filtration.</li>
	<li>Check under a light microscope to verify the removal of large debris and the intactness of the nuclear membrane. Nuclei need to be round and the nuclear membrane should not be distorted.</li>
	<li>Count nuclei using Trypan blue staining on a hemocytometer and aliquot nuclei for snRNA-seq/snATAC-seq.</li>
</ol>
5. Single nuclei RNA and ATAC seq
<ol>
	<li>Immediately process the nuclei using the single cell gene expression and single cell ATAC reagent kits (Table of Materials). 
	NOTE: The nuclei sample concentrations for the 10x Genomics system are 1500-3000 nuclei per &#181;L for snRNA-seq, and 3500-7000 nuclei per m &#181;L for snATAC-seq. The nuclei can be diluted using 1x PBS.</li>
	<li>Sequence the resulting libraries at the Genomics Core Facility.</li>
	<li>Perform quality control analysis of the data. Nuclei are included for further analysis if they contain Unique Molecular Identifier (UMI) &#62;1000, number of genes &#62;500 and percent of mitochondrial genes &#60;5%, and are within mean + three standard deviations of the UMIs and genes.</li>
</ol>

<ol><li>Huse, J. T., Holland, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+brain+cancer%3A+advances+in+the+molecular+pathology+of+malignant+glioma+and+medulloblastoma%5BTitle%5D)+AND+%22Nature+Reviews+Cancer%22%5BJournal%5D)">Targeting brain cancer: advances in the molecular pathology of malignant glioma and medulloblastoma.</a> Nature Reviews Cancer. 10 (5), 319-331 (2010).</li>
<li>Kreso, A., Dick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evolution+of+the+cancer+stem+cell+model%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Evolution of the cancer stem cell model.</a> Cell Stem Cell. 14 (3), 275-291 (2014).</li>
<li>Filbin, M. G., Suva, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gliomas+Genomics+and+Epigenomics%3A+Arriving+at+the+Start+and+Knowing+It+for+the+First+Time%5BTitle%5D)+AND+%22Annual+Review+of+Pathology%22%5BJournal%5D)">Gliomas Genomics and Epigenomics: Arriving at the Start and Knowing It for the First Time.</a> Annual Review of Pathology. 11, 497-521 (2016).</li>
<li>Ferris, S. P., Hofmann, J. W., Solomon, D. A., Perry, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+gliomas%3A+from+morphology+to+molecules%5BTitle%5D)+AND+%22Virchows+Archive%22%5BJournal%5D)">Characterization of gliomas: from morphology to molecules.</a> Virchows Archive. 471 (2), 257-269 (2017).</li>
<li>Louis, D. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+2016+World+Health+Organization+Classification+of+Tumors+of+the+Central+Nervous+System%3A+a+summary%5BTitle%5D)+AND+%22Acta+Neuropathologica%22%5BJournal%5D)">The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary.</a> Acta Neuropathologica. 131 (6), 803-820 (2016).</li>
<li>Eckel-Passow, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glioma+Groups+Based+on+1p%2F19q%2C+IDH%2C+and+TERT+Promoter+Mutations+in+Tumors%5BTitle%5D)+AND+%22The+New+England+Journal+of+Medicine%22%5BJournal%5D)">Glioma Groups Based on 1p/19q, IDH, and TERT Promoter Mutations in Tumors.</a> The New England Journal of Medicine. 372 (26), 2499-2508 (2015).</li>
<li>Suzuki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mutational+landscape+and+clonal+architecture+in+grade+II+and+III+gliomas%5BTitle%5D)+AND+%22Nature+Genetics%22%5BJournal%5D)">Mutational landscape and clonal architecture in grade II and III gliomas.</a> Nature Genetics. 47 (5), 458-468 (2015).</li>
<li>Cancer Genome Atlas Research. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comprehensive%2C+Integrative+Genomic+Analysis+of+Diffuse+Lower-Grade+Gliomas%5BTitle%5D)+AND+%22The+New+England+Journal+of+Medicine%22%5BJournal%5D)">Comprehensive, Integrative Genomic Analysis of Diffuse Lower-Grade Gliomas.</a> The New England Journal of Medicine. 372 (26), 2481-2498 (2015).</li>
<li>Noushmehr, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+a+CpG+island+methylator+phenotype+that+defines+a+distinct+subgroup+of+glioma%5BTitle%5D)+AND+%22Cancer+Cell%22%5BJournal%5D)">Identification of a CpG island methylator phenotype that defines a distinct subgroup of glioma.</a> Cancer Cell. 17 (5), 510-522 (2010).</li>
<li>Yan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((IDH1+and+IDH2+mutations+in+gliomas%5BTitle%5D)+AND+%22The+New+England+Journal+of+Medicine%22%5BJournal%5D)">IDH1 and IDH2 mutations in gliomas.</a> The New England Journal of Medicine. 360 (8), 765-773 (2009).</li>
<li>Yan, H., Bigner, D. D., Velculescu, V., Parsons, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mutant+metabolic+enzymes+are+at+the+origin+of+gliomas%5BTitle%5D)+AND+%22Cancer+Research%22%5BJournal%5D)">Mutant metabolic enzymes are at the origin of gliomas.</a> Cancer Research. 69 (24), 9157-9159 (2009).</li>
<li>Gawad, C., Koh, W., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+genome+sequencing%3A+current+state+of+the+science%5BTitle%5D)+AND+%22Nature+Reviews+Genetics%22%5BJournal%5D)">Single-cell genome sequencing: current state of the science.</a> Nature Reviews Genetics. 17 (3), 175-188 (2016).</li>
<li>Tanay, A., Regev, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Scaling+single-cell+genomics+from+phenomenology+to+mechanism%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Scaling single-cell genomics from phenomenology to mechanism.</a> Nature. 541 (7637), 331-338 (2017).</li>
<li>Wu, M., Singh, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microfluidic+Flow+Cytometry+for+Single-Cell+Protein+Analysis%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Microfluidic Flow Cytometry for Single-Cell Protein Analysis.</a> Methods in Molecular Biology. 1346, 69-83 (2015).</li>
<li>Schwartzman, O., Tanay, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+epigenomics%3A+techniques+and+emerging+applications%5BTitle%5D)+AND+%22Nature+Reviews+Genetics%22%5BJournal%5D)">Single-cell epigenomics: techniques and emerging applications.</a> Nature Reviews Genetics. 16 (12), 716-726 (2015).</li>
<li>Macaulay, I. C., Ponting, C. P., Voet, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-Cell+Multiomics%3A+Multiple+Measurements+from+Single+Cells%5BTitle%5D)+AND+%22Trends+in+Genetics%22%5BJournal%5D)">Single-Cell Multiomics: Multiple Measurements from Single Cells.</a> Trends in Genetics. 33 (2), 155-168 (2017).</li>
<li>Buenrostro, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+chromatin+accessibility+reveals+principles+of+regulatory+variation%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Single-cell chromatin accessibility reveals principles of regulatory variation.</a> Nature. 523 (7561), 486-490 (2015).</li>
<li>Habib, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Massively+parallel+single-nucleus+RNA-seq+with+DroNc-seq%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Massively parallel single-nucleus RNA-seq with DroNc-seq.</a> Nature Methods. 14 (10), 955-958 (2017).</li>
<li>Corces, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+improved+ATAC-seq+protocol+reduces+background+and+enables+interrogation+of+frozen+tissues%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues.</a> Nature Methods. 14 (10), 959-962 (2017).</li>
<li>Slyper, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+single-cell+and+single-nucleus+RNA-Seq+toolbox+for+fresh+and+frozen+human+tumors%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors.</a> Nature Medicine. 26 (5), 792-802 (2020).</li>
<li>Mathys, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+transcriptomic+analysis+of+Alzheimer%27s+disease%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Single-cell transcriptomic analysis of Alzheimer's disease.</a> Nature. 570 (7761), 332-337 (2019).</li>
<li>Krishnaswami, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Using+single+nuclei+for+RNA-seq+to+capture+the+transcriptome+of+postmortem+neurons%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons.</a> Nature Protocols. 11 (3), 499-524 (2016).</li>
<li>Al-Dalahmah, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-nucleus+RNA-seq+identifies+Huntington+disease+astrocyte+states%5BTitle%5D)+AND+%22Acta+Neuropathologica+Communications%22%5BJournal%5D)">Single-nucleus RNA-seq identifies Huntington disease astrocyte states.</a> Acta Neuropathologica Communications. 8 (1), 19(2020).</li>
<li>Jakel, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Altered+human+oligodendrocyte+heterogeneity+in+multiple+sclerosis%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Altered human oligodendrocyte heterogeneity in multiple sclerosis.</a> Nature. 566 (7745), 543-547 (2019).</li>
<li>Butler, A., Hoffman, P., Smibert, P., Papalexi, E., Satija, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrating+single-cell+transcriptomic+data+across+different+conditions%2C+technologies%2C+and+species%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">Integrating single-cell transcriptomic data across different conditions, technologies, and species.</a> Nature Biotechnology. , (2018).</li>
<li>Lake, B. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrative+single-cell+analysis+of+transcriptional+and+epigenetic+states+in+the+human+adult+brain%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">Integrative single-cell analysis of transcriptional and epigenetic states in the human adult brain.</a> Nature Biotechnology. 36 (1), 70-80 (2018).</li>
<li>Stuart, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comprehensive+Integration+of+Single-Cell+Data%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Comprehensive Integration of Single-Cell Data.</a> Cell. 177 (7), 1888-1902 (2019).</li>
<li>Bready, D., Placantonakis, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+Pathogenesis+of+Low-Grade+Glioma%5BTitle%5D)+AND+%22Neurosurgery+Clinics+of+North+America%22%5BJournal%5D)">Molecular Pathogenesis of Low-Grade Glioma.</a> Neurosurgery Clinics of North America. 30 (1), 17-25 (2019).</li>
<li>Venteicher, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decoupling+genetics%2C+lineages%2C+and+microenvironment+in+IDH-mutant+gliomas+by+single-cell+RNA-seq%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Decoupling genetics, lineages, and microenvironment in IDH-mutant gliomas by single-cell RNA-seq.</a> Science. 355 (6332), (2017).</li>
<li>Tirosh, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+RNA-seq+supports+a+developmental+hierarchy+in+human+oligodendroglioma%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Single-cell RNA-seq supports a developmental hierarchy in human oligodendroglioma.</a> Nature. 539 (7628), 309-313 (2016).</li>
<li>Weng, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-Cell+Transcriptomics+Uncovers+Glial+Progenitor+Diversity+and+Cell+Fate+Determinants+during+Development+and+Gliomagenesis%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Single-Cell Transcriptomics Uncovers Glial Progenitor Diversity and Cell Fate Determinants during Development and Gliomagenesis.</a> Cell Stem Cell. 24 (5), 707-723 (2019).</li>
<li>Neftel, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+Integrative+Model+of+Cellular+States%2C+Plasticity%2C+and+Genetics+for+Glioblastoma%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">An Integrative Model of Cellular States, Plasticity, and Genetics for Glioblastoma.</a> Cell. 178 (4), 835-849 (2019).</li>
<li>Al-Ali, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-nucleus+chromatin+accessibility+reveals+intratumoral+epigenetic+heterogeneity+in+IDH1+mutant+gliomas%5BTitle%5D)+AND+%22Acta+Neuropathologica+Communications%22%5BJournal%5D)">Single-nucleus chromatin accessibility reveals intratumoral epigenetic heterogeneity in IDH1 mutant gliomas.</a> Acta Neuropathologica Communications. 7 (1), 201(2019).</li>
</ol>

The authors have nothing to disclose.

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>CaCl&lt;sub&gt;2&lt;/sub&gt;</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 15 mL Conical Centrifuge Tubes</td><td>Fisher Scientific</td><td>352096</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 &amp;micro;m)</td><td>Miltenyi Biotec</td><td>130-098-458</td><td></td></tr><tr><td>MACS SmartStrainers (100 &amp;micro;m)</td><td>Miltenyi Biotec</td><td>130-098-463</td><td></td></tr><tr><td>Mg(Ac)&lt;sub&gt;2&lt;/sub&gt;</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 &amp;micro;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 &amp;micro;L)</td><td>Themo Fisher Scientific</td><td>2079GPK</td><td></td></tr><tr><td>Wide Bore pipette tips (200 &amp;micro;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

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 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 filtration...

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 ...

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Research

JoVE Journal

Cancer Research

This video describes the protocol to isolate single-nuclei from fresh frozen glioma tissue. The isolated single-nuclei can be used as specimens for sequencing techniques such as single-nuclei RNA (snRNA) sequencing and single-nuclei Assay for Transposase-Accessible Chromatin (snATAC) sequencing to obtain deeper insights into inter- and intra-tumor heterogeneity observed in glioma of the central nervous tissues.

Isolation of Nuclei from Fresh Frozen Glioma Tissues: A Method to Obtain Intact Nuclei from Glioma Tumor Samples

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Single nuclei isolation relies on dissociation and detergent-based permeabilization of the cell membrane, steps that need optimization and are prone to introducing technical artifacts. We demonstrate a detergent and enzyme-free protocol for rapid isolation of intact nuclei directly from whole tissue, yielding nuclei suitable for single-nucleus RNA-seq (snRNA-Seq) or ATAC-seq.

Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method

We have developed a method that enriches for and isolates human astrocyte populations from fresh-frozen tissue for use in downstream molecular analyses.

Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

Here, we present a protocol for isolating nuclei from flash-frozen, archived liver tissues for single-nucleus RNA-seq, ATAC-seq, and joint multiomics (RNA-seq and ATAC-seq).

Isolation of Nuclei from Flash-Frozen Liver Tissue for Single-Cell Multiomics

Here, we present a protocol describing cell nuclei preparation. After microdissection and enzymatic dissociation of cardiac tissue into single cells, the progenitor cells were frozen, followed by isolation of pure viable cells, which were used for single-nucleus RNA sequencing and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing analyses.

Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

The study describes a simple, quick, and partially automated protocol to isolate high-quality nuclei from frozen mammalian tissues for downstream single nuclei RNA sequencing.

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

The success of single-cell/single-nuclei transcriptomics and multi-omics largely depends on the quality of cells/nuclei. Therefore, isolating cells/nuclei from tissue and their purification must be highly standardized. This protocol describes the preparation of nuclei from the brain and bone marrow for downstream single-nuclei multiome assay.

High-Quality Brain and Bone Marrow Nuclei Preparation for Single Nuclei Multiome Assays

The protocol provides a reliable and optimized approach to the isolation of nuclei from solid tumor specimens for multiome sequencing using the 10x Genomics platform, including recommendations for tissue dissociation conditions, cryopreservation of single-cell suspensions, and assessment of isolated nuclei.

Optimized Nuclei Isolation from Fresh and Frozen Solid Tumor Specimens for Multiome Sequencing

Here, we introduce a comprehensive protocol for the generation and downstream analysis of human brain organoids using single-cell and single-nucleus RNA sequencing.

Generation and Downstream Analysis of Single-Cell and Single-Nuclei Transcriptomes in Brain Organoids

Learn nuclei isolation from fresh frozen brain tumors for single-nucleus RNA-seq and ATAC-seq using gradient centrifugation. This efficient method yields high-quality nuclei for studying tumor heterogeneity and evolution.

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

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