我们感谢德国癌症中心 （DKFZ） 的单细胞开放实验室 （scOpenLab） 的有益讨论。这项研究得到了德国癌症援助组织马克斯-埃德计划赠款编号70111964（S.T.）的支持。

新鲜冷冻胶质瘤样本来自德国海德堡国家肿瘤疾病中心（NCT）组织库。海德堡医学院机构审查委员会批准了患者材料的使用，并获得了研究中包括的所有患者的知情同意。
1. 实验准备
<ol><li>在冰上或在 4 °C 下执行所有步骤。</li>
	<li>预冷管、盘子、剃须刀刀片、杜恩克和害虫至 4 °C。</li>
	<li>提前准备所有缓冲。这些缓冲器在室温下是稳定的。建议进行无菌过滤，尤其是蔗糖。股票准备从科塞斯等人19日修改。请参阅表 1-7。</li>
	<li>从液氮或 -80 °C 冰柜存储中取出样品，并保持干燥的冰层，直到第 2.1 步。</li>
</ol>2. 组织解剖和分离
<ol><li>将新鲜冷冻组织样本（10-60毫克）转移到预冷的培养皿中。薄荷/切碎新鲜冷冻组织，用剃须刀刀片切成冰上的小块。</li>
	<li>在预冷的 1.5 mL 管中加入 500 μL 的冰镇核裂解缓冲器。将组织片放在含有核裂解缓冲器的1.5mL管中，并转移到杜塞尔（材料表）。 注:有两个 Dounce 同质化害虫:初始样品减少的"松散"或"A"害虫和完全样本均质化的"紧"或"B"害虫。</li>
	<li>将组织片与"松散"的害虫缠成约20次，直到摩擦减少。如果存在碎屑，则样品可以通过 100 mm 滤网过滤预先清除。</li>
	<li>Dounce 与 "紧" 害虫 20 笔触， 以实现完整的组织均质化。</li>
	<li>将同质性（约 500 μL）转移到预冷的 2 mL 管中。添加 1 mL 的冰镇裂解缓冲器。轻轻混合，在冰上孵育5分钟。轻轻地与宽孔移液器尖端混合，在孵化过程中重复 1-2 次。</li>
	<li>使用 30 μm 滤网过滤整个同质性，收集到 15 mL 猎鹰管中，然后传输回新的预冷 2 mL 管中。单个过滤器通常足以满足整个同质性。</li>
	<li>在光显微镜下检查，以验证大碎片的清除和核膜的完整性。核需要圆，核膜不应该扭曲。如果存在碎片，可以重新过滤原子核。</li>
	<li>在长凳顶部离心机上，将核离心在 500 x g 下 5 分钟，在 4 °C 下离心。取出超高纳坦，留下 +50 μL，颗粒中含有核。轻轻地将颗粒重新吸回另一个 1 mL 的核裂解缓冲器中，并在冰上孵育 5 分钟。</li>
	<li>在 4°C 下将核离心在 500 x g 下 5 分钟。 去除超高颗粒而不打扰颗粒，加入500 mL的1倍同质化缓冲（HB）（表4），孵育5分钟，无需再悬浮。然后，将核重新悬回另一个 1.0 mL 的 1x HB 中。</li>
	<li>在 4 °C 下将核离心在 500 x g 下 5 分钟。 取出超高纳坦，轻轻将 1x HB 的 200 μL 内的核重新插入新的 2.0 mL 管中。</li>
</ol>3. 梯度离心
<ol><li>加入200微升50%碘化醇溶液（表5），最终浓度为25%碘化醇。混合好10次，移液器设置在300ml。</li>
	<li>在 25% 混合物下加入 300 μL 的 29% 碘化醇溶液 （表 6）。使用 P1000 精细尖端以避免层的混合物。</li>
	<li>在 29% 混合物下加入 300 μl 的 35% 碘化醇溶液 （表 7）。使用 P1000 精细尖端以避免层的混合物。 注意:此步骤要求在管道过程中逐步去除移液器尖端，以避免过多的体积位移。</li>
	<li>将样品放在摆动的桶离心机中，在 4°C 下以 3，500 x g 的速度旋转 20 分钟，同时关闭制动器。</li>
	<li>轻轻取出样品，不摇晃，在光线下观察。在第二层和第三层之间应可见 95% 纯核的清晰白色波段（图 1）。</li>
</ol>4. 核分离
<ol><li>以 29%-35% 的相间水平吸气顶层，直到白核带。</li>
	<li>以 200 mL 的体积收集核带，转移到新鲜管中，并用 20μm 滤芯过滤器（材料表）进行过滤。 注:核不需要在过滤前重新悬浮。</li>
	<li>在光显微镜下检查，以验证大碎片的清除和核膜的完整性。核需要圆，核膜不应该扭曲。</li>
	<li>计数核使用特里潘蓝色染色在六细胞计和阿利库特核为 snrna - seq / snatac - seq 。</li>
</ol>5. 单核RNA和ATAC seq
<ol><li>立即使用单细胞基因表达和单细胞ATAC试剂盒（材料表）处理核。 注:10 倍基因组系统的核样本浓度为 snRNA-seq 的 1500-3000 个核，snNA-seq 的核样本浓度为 3500-7000 个核/微升。使用 1x PBS 可以稀释核。</li>
	<li>在基因组核心设施对生成的库进行排序。</li>
	<li>对数据进行质量控制分析。如果核含有独特的分子识别器 （UMI） >1000，则包含 500 >基因数量和线粒体基因的百分比<5%，并且均值在 UMI 和基因的三个标准偏差范围内，则包括核进行进一步分析。</li>
</ol>

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作者没有什么可透露的。

肿瘤内异质性领域正处于一个激动人心的阶段，正在开发新的分析和平台来挑战和扩展现有的知识。肿瘤内异质性是导致胶质瘤28中疾病进展和抵抗当前治疗方式的关键因素。最近关于脑肿瘤的研究集中在这一重要方面，使用单细胞转录和表观基因组测定，以更好地描述细胞异质性在同一肿瘤29，30，31，32。<s...

扩散性低级胶质瘤（LGG）是成人中最常见的原发性脑肿瘤，是脑半球中经常出现的渗透性肿瘤。LGG表现出肿瘤间和肿瘤内异质性，这不仅是由肿瘤人群驱动的，而且是由复杂参与肿瘤发育和进展的非恶性细胞驱动的，1、2、3、4、5。
在过去十年中，在胶质瘤领域收集的基因组数据大量增加。这些数据主要来自散装肿瘤测序研究，对分子定性以及目前脑肿瘤5、6、7、8、9、10、11的分类做出了巨大贡献。然而，尽管这些研究揭示了与胶质瘤相关的广泛的分子景观，但在治疗干预方面仍然缺乏进展，令人失望。脑肿瘤治疗耐药性的障碍之一是肿瘤内异质性。为了解决这个问题，各种研究一直集中在基因组学，转录学，蛋白体学和表观遗传异质性存在于肿瘤的单细胞水平12，13，14，15，16，17。
虽然在过去几年中，单细胞领域取得了显著的技术进步，但主要限制因素之一是提供分离细胞和进行这些实验所需的新鲜标本。为了克服这一限制，已经进行了几次成功的尝试，例如从冷冻组织中，使用核而不是细胞18，19进行snRNA-seq和snATAC-seq。这些方法大多依赖于荧光激活的细胞分拣 （FACS） 或过滤策略。单细胞和单核方法都有其优点和缺点。单细胞方法保持线粒体记录，虽然可能信息丰富，但也可能减少转录体覆盖，因为他们的高丰度。单核分离方法消除了线粒体分数的高百分比，从而使核记录20的更深入的覆盖。
近年来，有各种商业平台被用来检测单细胞基因组学数据，包括RNA-seq和ATAC-seq。其中最突出的平台是10倍基因组铬平台，用于单细胞基因表达和单细胞ATAC分析。由于该平台在微流体室的帮助下工作，任何碎屑或聚合物都可能堵塞系统，导致数据、试剂和有价值的临床样本丢失。因此，单细胞研究的成功在很大程度上取决于单细胞/细胞核的准确分离。
我们将在这里展示的协议是DroNc-seq和Omni-ATAC-seq协议的稍微修改的组合，并利用类似的方法进行最近的研究，利用snRNA-seq来了解人脑中的神经紊乱和神经元细胞类型18，19，21，22，23，24。该协议采用冷冻样品的酶/机械分离组合，然后进行过滤和梯度离心，并允许从新鲜冷冻胶质瘤组织快速准确地分离单核。我们已经成功地利用这个协议从脑肿瘤标本的相同核制剂中生成snRNA-seq和snATAC-seq数据。

<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

成人扩散胶质瘤表现出肿瘤间和肿瘤内异质性。直到最近，大多数大规模的分子分析工作都集中在导致脑肿瘤分子分类的批量方法上。在过去五年中，单细胞测序方法突出了胶质瘤的几个重要特征。这些研究大多利用新鲜的脑肿瘤标本，利用流细胞学或抗体分离方法分离单细胞。今后，使用来自生物库的新鲜冷冻组织样本将为单细胞应用提供更大的灵活性。此外，随着单细胞场的进步，下一个挑战将是从单个细胞或相同的样本制备中生成多细胞数据，以更好地解开肿瘤的复杂性。因此，简单而灵活的协议，允许数据生成的各种方法，如单核RNA测序（snRNA-seq）和单核检测的转位酶-可访问色度素与高通量测序（snATAC-seq）将是重要的领域。
单细胞领域的最新进展，加上10倍基因组学平台等可访问的微流体仪器，促进了单细胞在研究实验室中的应用。为了研究脑肿瘤异质性，我们开发了一个增强的方案，用于将单核从新鲜冷冻胶质瘤中分离出来。此协议合并了现有的单细胞协议，并结合了同质化步骤，然后是过滤和缓冲介导梯度离心。生成的样本是纯单核悬浮，可用于从相同的核制剂生成单核基因表达和色度素可访问数据。

单核基因组学是一个不断发展的领域，数据和协议有限。影响单核检测结果的一个关键因素是纯核和完整核的分离。我们结合了两个已发布的协议（DroNc-seq 和 Omni-ATAC-seq 协议），在相对较短的时间内将高质量和纯核从新鲜冷冻胶质瘤组织块中分离出来，从而保持成绩单的稳定性（图 1）。
使用各种过滤步骤以及使用碘糖醇/蔗糖梯度的梯度离心，可以隔?...

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

肿瘤内异质性是肿瘤的内在特征，包括胶质瘤。我们开发了一个简单而高效的协议，利用缓冲和梯度离心的组合，将单核从新鲜冷冻胶质瘤组织中分离出来，用于单核RNA和ATAC测序研究。

单核RNA-seq和ATAC-seq的新鲜冷冻脑肿瘤的核隔离

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

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

Research

JoVE Journal

Cancer Research

12.0K 次观看.  University Hospital Heidelberg. 肿瘤内异质性是肿瘤的内在特征，包括胶质瘤。我们开发了一个简单而高效的协议，利用缓冲和梯度离心的组合，将单核从新鲜冷冻胶质瘤组织中分离出来，用于单核RNA和ATAC测序研究。

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

分离细胞核用于实体瘤标本中的多组测序

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

Multiome sequencing, which provides same-cell/paired single-cell RNA- and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) data, represents a breakthrough in our ability to discern tumor cell heterogeneity-a primary focus of translational cancer research at this time. However, the quality of sequencing data acquired using this advanced modality is highly dependent on the quality of the input material. 
Digestion conditions need to be optimized to maximize cell yield without sacrificing quality. This is particularly challenging in the context of solid tumors with dense desmoplastic matrices that must be gently broken down for cell release. Freshly isolated cells from solid tumor tissue are more fragile than those isolated from cell lines. Additionally, as the cell types isolated are heterogeneous, conditions should be selected to support the total cell population. 
Finally, nuclear isolation conditions must be optimized based on these qualities in terms of lysis times and reagent types/ratios. In this article, we describe our experience with nuclear isolation for the 10x Genomics multiome sequencing platform from solid tumor specimens. We provide recommendations for tissue digestion, storage of single-cell suspensions (if desired), and nuclear isolation and assessment.

作者聚焦：在具有挑战性的肿瘤微环境中增强用于多组测序的细胞核分离

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.

从新鲜和冷冻实体瘤标本中优化细胞核分离，用于多组学测序

Multiome sequencing, which provides same-cell/paired single-cell RNA- and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) ...

科研

癌症研究

以单细胞分辨率从小鼠心脏祖细胞中分离细胞核

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

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene expression and chromatin state of individual cells allows the identification of the cell type and state. Single-cell sequencing approaches have revealed a number of important characteristics of cardiac progenitor cell heterogeneity. However, these methods are generally restricted to fresh tissue, which limits studies with diverse experimental conditions, as the fresh tissue must be processed at once in the same run to reduce the technical variability. Therefore, easy and flexible procedures to produce data from methods such as single-nucleus RNA sequencing (snRNA-seq) and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing (snATAC-seq) are needed in this area. Here, we present a protocol to rapidly isolate nuclei for subsequent single-nuclei dual-omics (combined snRNA-seq and snATAC-seq). This method allows the isolation of nuclei from frozen samples of cardiac progenitor cells and can be combined with platforms that use microfluidic chambers.

作者聚焦:从小鼠心脏祖细胞中分离细胞核，用于单细胞分辨率下的表观基因组和基因表达分析  

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.

从小鼠心脏祖细胞中分离细胞核，用于单细胞分辨率的表观基因组和基因表达谱分析

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene ...

发育生物学

从人脑类器官中提取单细胞和细胞核

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

Over the past decade, single-cell transcriptomics has significantly evolved and become a standard laboratory method for simultaneous analysis of gene expression profiles of individual cells, allowing the capture of cellular diversity. In order to overcome limitations posed by difficult-to-isolate cell types, an alternative approach aiming at recovering single nuclei instead of intact cells can be utilized for sequencing, making transcriptome profiling of individual cells universally applicable. These techniques have become a cornerstone in the study of brain organoids, establishing them as models of the developing human brain. Leveraging the potential of single-cell and single-nucleus transcriptomics in brain organoid research, this protocol presents a step-by-step guide encompassing key procedures such as organoid dissociation, single-cell or nuclei isolation, library preparation&#160;and sequencing. By implementing these alternative approaches, researchers can obtain high-quality datasets, enabling the identification of neuronal and non-neuronal cell types, gene expression profiles, and cell lineage trajectories. This facilitates comprehensive investigations into cellular processes and molecular mechanisms shaping brain development.

作者聚焦:将类器官模型与单细胞和空间转录组学技术相结合

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

脑类器官中单细胞和单核转录组的生成和下游分析

Over the past decade, single-cell transcriptomics has significantly evolved and become a standard laboratory method for simultaneous analysis of gene ...

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

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

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.

成人脊髓细胞核在大规模平行单核 RNA 测序中的分离

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

单核RNA-seq和ATAC-seq的新鲜冷冻脑肿瘤的核隔离

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从新鲜和冷冻实体瘤标本中优化细胞核分离，用于多组学测序

从新鲜和冷冻实体瘤标本中优化细胞核分离，用于多组学测序

从新鲜和冷冻实体瘤标本中优化细胞核分离，用于多组学测序

从小鼠心脏祖细胞中分离细胞核，用于单细胞分辨率的表观基因组和基因表达谱分析

从小鼠心脏祖细胞中分离细胞核，用于单细胞分辨率的表观基因组和基因表达谱分析

从小鼠心脏祖细胞中分离细胞核，用于单细胞分辨率的表观基因组和基因表达谱分析

脑类器官中单细胞和单核转录组的生成和下游分析

脑类器官中单细胞和单核转录组的生成和下游分析

脑类器官中单细胞和单核转录组的生成和下游分析

成人脊髓细胞核在大规模平行单核 RNA 测序中的分离