我们要感谢斯坦福大学功能基因组学设施（SFGF），特别是Dhananjay Wagh和John Coller，以及10x Genomics在优化我们的实验方面的帮助。我们还要感谢 George Poultsides 博士、Monica Dua、Brendan Visser 博士和 Byrne Lee 博士在获取患者标本方面的帮助。我们要感谢 Art 和 Elaine Taylor、Rantz 基金会以及 Warren 和 Judy Kaplan 对我们研究工作的慷慨支持。资金来源包括 NIH 赠款 1F32CA23931201A1 （D.S.F.）、1R01GM116892 （M.T.L.）、1R01GM136659 （M.T.L.）、Goldman Sachs Foundation （J.A.N.， D.S.F.， M.T.L.）、Damon Runyon 癌症研究基金会 （D.D.， M.T.L.）、Gunn/Olivier 基金、加州再生医学研究所、Stinehart/Reed 基金会和 Hagey 儿科再生医学实验室。使用NIH资金购买的机器（S10OD025212、S10OD018220和1S10OD01058001A1）进行测序。

人类胰腺癌（胰腺导管腺癌）样本是根据 IRB 批准的方案在我们的实验室中采集的。获得患者组织采集的知情同意书。将组织从手术室运送到实验室，然后按如下方式处理。
1.组织解离（消化）
<ol><li>准备消化缓冲液（参见 材料表）。</li>
	<li>肿瘤切除后尽快获得感兴趣的组织，并将标本在淬灭缓冲液（含~5%胎牛血清的DMEM F-12）或冰上的1x PBS中运输。</li>
	<li>在90mm培养皿中，使用干净的镊子和剪刀在3-5mL消化缓冲液中切碎组织（图2A）。</li>
	<li>将切碎的组织转移到50mL锥形管中，并使用具有振荡功能的水浴或干搅拌器在~10mL消化缓冲液中解离30分钟。</li>
	<li>从搅拌器中取出，用~20 mL淬火培养基淬灭。通过100μm细胞过滤器将溶液过滤到干净的锥形管中。</li>
	<li>从过滤器中收集任何剩余的固体组织（如果需要，重新切碎剩余的组织块），并在37°C下再放入~10mL新鲜消化缓冲液中30分钟。重复直到所有肿瘤组织都解离。</li>
	<li>将池细胞悬浮液等分试样并通过 70 μm，然后用 40 μm 细胞过滤器过滤到冰上的干净锥形管中。</li>
	<li>在4°C下离心至500× g 沉淀细胞5分钟，然后倾析上清液。</li>
</ol>2. 冷冻保存
<ol><li>通过将沉淀重悬于 1 mL 1x PBS 中来冲洗细胞。</li>
	<li>将细胞悬液（~1-1000 万个细胞/管以获得最佳冷冻效果）转移到冰上的 1.5 mL 冻存管中。</li>
	<li>在4°C下离心至500× g 沉淀细胞5分钟，然后倾析上清液。</li>
	<li>将细胞重悬于1mL Bambanker溶液中，转移到1.5或2mL冷冻管中（图2B），并在-80°C下使用-1°C / min冷却速率冷冻容器（含有100%异丙醇），如果预计样品长期储存，则转移到液氮中。</li>
</ol>3. 细胞核分离
<ol><li>快速解冻细胞悬液的冷冻管并将其放在湿冰上。</li>
	<li>将解冻的细胞悬浮液转移到 2 mL 微量离心管中，并将小瓶加满 2 mL 溶液中，加入 1x PBS 冲洗细胞。</li>
	<li>使用血细胞计数器获得手动细胞计数，以评估细胞的数量和质量（图2C）。</li>
	<li>在4°C下离心至500× g 沉淀细胞5分钟，然后使用逐渐变小的移液器吸头轻轻倾析上清液，留下干燥的沉淀并将其保持在冰上。<ol><li>所有离心步骤均使用旋转桶转子。为了获得最大的细胞产量，将试管放入离心机中，铰链朝外，然后用移液器尖端朝向试管的另一侧进行卧螺离心（图2D）。 注意：对于倾析上清液，请使用逐渐变小的移液器吸头去除液体，以限制沉淀的破坏，同时最大限度地倾析液体的体积。这种策略在细胞核提取后的方案后期特别有用，因为细胞核颗粒通常是不可见的。</li>
	</ol></li>
	<li>根据已发表的方案制备1x细胞裂解缓冲液，细胞裂解稀释缓冲液和洗涤缓冲液（图3A），并进行以下修改（参见 表1，材料表）： 注意：在使用之前，将洋地黄皂苷放在65°C的加热块上以溶解沉淀物（图3B）。这通常需要大约 10 分钟。</li>
	<li>将 100 μL 1x 细胞裂解缓冲液和 900 μL 细胞裂解稀释缓冲液混合，轻轻移液混合，然后将其置于冰上，制备 0.1x 细胞裂解缓冲液。</li>
	<li>将细胞沉淀重悬于100μL冷却的0.1x细胞裂解缓冲液中，轻轻移液至混合5x。</li>
	<li>在冰上孵育3分钟（图3C）。</li>
	<li>加入 1 mL 冷冻洗涤缓冲液，上下移液，轻轻混合 5 倍。</li>
	<li>离心使细胞核在4°C下以500× g 沉淀5分钟，轻轻将上清液倾倒至干燥的沉淀中，并保持在冰上。 注意：如果起始细胞数量较低（100,000-200,000个细胞），则在200μL PCR管而不是2mL微量离心管中执行细胞裂解和洗涤步骤，以减少管表面积并最大限度地减少损失。在这种情况下，请调低洗涤缓冲液体积以适应较小的试管体积。</li>
	<li>重复步骤 3.9-3.10 2 次，共洗涤 3 次。</li>
	<li>在显微镜下使用血细胞计数器载玻片手动计数细胞核。如果需要，请使用台盼蓝染料，以提供对比度以观察细胞核，并帮助区分细胞核和未裂解的细胞。</li>
	<li>根据公布的方案制备 1x 细胞核缓冲液：20x 细胞核缓冲液原液、1 mM DTT、1 U/μL RNase 抑制剂在无核酸酶水中并保持在冰上（参见 材料表）。</li>
	<li>根据目标靶向细胞核回收率，将细胞核沉淀重悬于1x细胞核缓冲液中。 注意：如果浓度足够高，则在确定起始重悬体积时，在确定起始重悬体积时，目标回收率为10,000或略高（3,230-8,060核/ μL），因为预期的体积损失为25μL，过滤时浓度降低20%（步骤3.15）。</li>
	<li>轻轻地将重悬的细胞核体积通过40μm移液器尖端细胞过滤器，并将细胞核放在冰上（图3D）。</li>
	<li>重新计数细胞核（图4A-C）。如果需要，用Nuclei Buffer进一步稀释最终溶液。</li>
	<li>在冰上运输细胞核，并立即按照公布的方案进行细胞核捕获。</li>
</ol>

分离细胞核用于实体瘤标本中的多组测序

<ol>
	<li>Jain, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+RNA+sequencing+and+spatial+transcriptomics+reveal+cancer-associated+fibroblasts+in+glioblastoma+with+protumoral+effects.">Single-cell RNA sequencing and spatial transcriptomics reveal cancer-associated fibroblasts in glioblastoma with protumoral effects.</a> J Clin Invest. 133 (5), e147087 (2023).</li><li>Sahai, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+framework+for+advancing+our+understanding+of+cancer-associated+fibroblasts.">A framework for advancing our understanding of cancer-associated fibroblasts.</a> Nat Rev Cancer. 20 (3), 174-186 (2020).</li><li>Foster, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiomic+analysis+reveals+conservation+of+cancer-associated+fibroblast+phenotypes+across+species+and+tissue+of+origin.">Multiomic analysis reveals conservation of cancer-associated fibroblast phenotypes across species and tissue of origin.</a> Cancer Cell. 40 (11), 1392-1406 (2022).</li><li>Hasan, S., Buechler, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What's+in+a+name?+An+emerging+framework+for+cancer-associated+fibroblasts,+myofibroblasts,+and+fibroblasts.">What's in a name? An emerging framework for cancer-associated fibroblasts, myofibroblasts, and fibroblasts.</a> Cancer Cell. 40 (11), 1273-1275 (2022).</li><li>. Nuclei isolation from complex tissues for single cell multiome ATAC + gene expression sequencing Available from: <a target="_blank" href="https://www.10xgenomics.com/support/single-cell-multiome-atac-plus-gene-expression/documentation/steps/sample-prep/nuclei-isolation-from-complex-tissues-for-single-cell-multiome-atac-plus-gene-expression-sequencing">https://www.10xgenomics.com/support/single-cell-multiome-atac-plus-gene-expression/documentation/steps/sample-prep/nuclei-isolation-from-complex-tissues-for-single-cell-multiome-atac-plus-gene-expression-sequencing</a> (2022)</li><li>. Nuclei isolation from embryonic mouse brain tissue for single cell multiome ATAC + gene expression sequencing Available from: <a target="_blank" href="https://www.10xgenomics.com/support/single-cell-multiome-atac-plus-gene-expression/documentation/steps/sample-prep/nuclei-isolation-from-embryonic-mouse-brain-tissue-for-single-cell-multiome-atac-plus-gene-expression-sequencing">https://www.10xgenomics.com/support/single-cell-multiome-atac-plus-gene-expression/documentation/steps/sample-prep/nuclei-isolation-from-embryonic-mouse-brain-tissue-for-single-cell-multiome-atac-plus-gene-expression-sequencing</a> (2022)</li><li>Januszyk, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+diabetic+and+non-diabetic+foot+ulcers+using+single-cell+RNA-sequencing.">Characterization of diabetic and non-diabetic foot ulcers using single-cell RNA-sequencing.</a> Micromachines (Basel). 11 (9), 815 (2020).</li><li>Foster, D. S., Jones, R. E., Ransom, R. C., Longaker, M. T., Norton, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolving+relationship+of+wound+healing+and+tumor+stroma.">The evolving relationship of wound healing and tumor stroma.</a> JCI Insight. 3 (18), e99911 (2018).</li><li>Nott, A., Schlachetzki, J. C. M., Fixsen, B. R., Glass, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclei+isolation+of+multiple+brain+cell+types+for+omics+interrogation.">Nuclei isolation of multiple brain cell types for omics interrogation.</a> Nat Protoc. 16 (3), 1629-1646 (2021).</li><li>Foster, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+spatial+multiomics+reveals+fibroblast+fate+during+tissue+repair.">Integrated spatial multiomics reveals fibroblast fate during tissue repair.</a> Proc Natl Acad Sci U S A. 118 (41), e2110025118 (2021).</li><li>Leiz, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclei+isolation+from+adult+mouse+kidney+for+single-nucleus+RNA-sequencing.">Nuclei isolation from adult mouse kidney for single-nucleus RNA-sequencing.</a> J Vis Exp. (175), (2021).</li><li>Narayanan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.</a> J Vis Exp. (162), e61542 (2020).</li></ol>

作者没有需要披露的利益冲突。

解开肿瘤微环境中存在的异质细胞群是癌症生物学的一个活跃关注领域。同样，复杂组织存在于良性病理中，例如伤口愈合和纤维化。多组测序已成为一种强大的工具，可以获取同细胞配对的scRNA和ATAC-seq数据。该协议描述了细胞核的分离，这需要在处理新鲜，脆弱，小肿瘤标本的环境中进行优化。在这里，我们提供了从结缔组织增生性实体瘤组织标本中分离细胞核的方案。我们发现这种方法即使...

随着我们对肿瘤生物学知识的增长，分析肿瘤微环境中异质细胞的重要性也有所增加 1,2。以配对细胞方式（多组测序）从同一细胞获取单细胞 RNA 和使用测序（ATAC 测序）数据检测转座酶可及染色质的能力为实现这一目标提供了重大进展 3,4。然而，这些实验既昂贵又耗时，所获得数据的质量和影响在很大程度上取决于实验条件和材料的质量。细胞核分离的标准化方案已发表 5,6。新鲜和异质组织需要方案优化，因为从实体瘤标本中新鲜分离的细胞比从细胞系中分离的细胞更脆弱。
另一个考虑因素是，对于实体瘤，手术标本通常要到当天晚些时候才能从手术室获得。因此，在没有冷冻保存步骤的情况下，直接从样品采集到细胞核捕获通常是不可行的。根据我们的经验，冷冻单细胞悬浮液可产生最高质量的细胞核（而不是快速冷冻的整个组织或其他保存方式）。对于具有高RNase含量的酶组织类型（如胰腺）尤其如此。
还需要设计组织消化条件，以便在不牺牲质量的情况下最大限度地提高细胞产量7。在具有致密结缔组织增生基质的实体瘤类型的背景下 8，细胞外基质必须被轻轻分解以释放细胞。此外，由于分离的细胞类型是异质的，因此应调整条件以支持总细胞群。人胰腺癌（胰腺导管腺癌）样品用于所述方案。胰腺癌是一种高度结缔组织增生的肿瘤类型，预示着相对粘稠的组织和细胞。此外，由于可用于研究的胰腺肿瘤标本也往往相对较小，因此努力最大限度地捕获细胞数量。
细胞核的分离需要在细胞裂解条件和时间以及试剂类型和比例方面进行最优化。在分离过程中处理细胞核也需要非常小心。在本文中，我们描述了我们从实体瘤组织中优化 10x Genomics 多组测序平台的核分离的经验（图 1）。我们提供组织消化、单细胞悬浮液冷冻保存（如果需要）和细胞核分离的建议。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100, 70, and 40 &#956;m Falcon cell strainers&#160;</td>
			<td>&#160;ThermoFisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10x Genomics Nuclei Buffer (20x)</td>
			<td>10x Genomics</td>
			<td>2000153/2000207</td>
			<td></td>
		</tr>
		<tr>
			<td>Bambanker&#160;</td>
			<td>Wako, Fisher Scientitic</td>
			<td>NC9582225&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-376</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>499609</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase (Collagenase Type IV)</td>
			<td>ThermoFisher</td>
			<td>17104019</td>
			<td></td>
		</tr>
		<tr>
			<td>Digitonin</td>
			<td>Thermo Fisher</td>
			<td>BN2006</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Worthington</td>
			<td>LS006330</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma Aldrich</td>
			<td>646563</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium F-12</td>
			<td>Thermo Fisher</td>
			<td>11320082</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher</td>
			<td>10438026</td>
			<td></td>
		</tr>
		<tr>
			<td>Flowmi 40 &#956;m&#160; Pipette Tip Cell Strainer&#160;</td>
			<td>Sigma Aldrich</td>
			<td>BAH136800040</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Histopaque-1119 Gradient Cell Separation solution</td>
			<td>Sigma Aldrich</td>
			<td>11191</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Sigma Aldrich</td>
			<td>M2520</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma Aldrich</td>
			<td>M1028</td>
			<td></td>
		</tr>
		<tr>
			<td>Miltenyi GentleMACSTM digest kit&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma Aldrich</td>
			<td>59222C</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Cryo &#34;Mr. Frosty&#34; Freezing Container</td>
			<td>&#160;ThermoFisher</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonident P40 Substitute</td>
			<td>Sigma Aldrich</td>
			<td>74385</td>
			<td></td>
		</tr>
		<tr>
			<td>Poloxamer 188</td>
			<td>Sigma</td>
			<td>P5556</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase inhibitor&#160;</td>
			<td>Sigma Aldrich</td>
			<td>3335399001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma Aldrich</td>
			<td>T2194</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Thermo Fisher</td>
			<td>85113</td>
			<td></td>
		</tr>
	</tbody>
</table>

optimized nuclei isolation from fresh and frozen solid tumor specimens for multiome sequencing

多组测序提供同细胞/配对的单细胞 RNA，以及具有测序（ATAC 测序）数据的转座酶可及染色质测定，代表了我们辨别肿瘤细胞异质性的能力的突破——这是目前转化癌症研究的主要焦点。然而，使用这种先进方式获取的测序数据的质量在很大程度上取决于输入材料的质量。 
需要优化酶切条件，以在不牺牲质量的情况下最大限度地提高细胞产量。这在具有致密结缔组织增生基质的实体瘤中尤其具有挑战性，这些基质必须被轻轻分解才能释放细胞。从实体瘤组织中新鲜分离的细胞比从细胞系中分离的细胞更脆弱。此外，由于分离的细胞类型是异质的，因此应选择条件来支持总细胞群。 
最后，必须根据裂解时间和试剂类型/比例等这些特性优化核分离条件。在本文中，我们描述了我们在 10x Genomics 多组测序平台从实体瘤标本中进行核分离的经验。我们提供组织消化、单细胞悬浮液储存（如果需要）以及细胞核分离和评估的建议。

As our knowledge of tumor biology grows, the importance of analyzing heterogeneous cells across the tumor microenvironment has also increased1,2. The ability to acquire single-cell RNA and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) data from the same cell in a paired-cell fashion (multiome sequencing) provides a significant advance towards this end3,4. These experiments are expensive and time-consuming, however, and the quality and impact of the data acquired are highly dependent on the quality of the experimental conditions and materials. Standardized protocols for nuclei isolation have been published5,6. Fresh and heterogeneous tissues require protocol optimization since freshly isolated cells from solid tumor specimens are more fragile than those isolated from cell lines.
Another consideration is that for solid tumors, surgical specimens are often not available from the operating room until late in the day. As such, it is generally not feasible to proceed directly from sample acquisition to nuclei capture without a cryopreservation step. In our experience, freezing a single-cell suspension yields the highest-quality nuclei (rather than flash-frozen whole tissue or other modalities of preservation). This is particularly true for enzymatic tissue types with high RNase content such as the pancreas.
Tissue digestion conditions also need to be designed to maximize cell yield without sacrificing quality7. In the context of solid tumor types with dense desmoplastic matrices8, the extracellular matrix must be gently broken down for cell release. Additionally, because the cell types isolated are heterogeneous, conditions should be adjusted to support the total cell population. Human pancreatic cancer (pancreatic ductal adenocarcinoma) samples are used in the described protocol. Pancreatic cancer represents a highly desmoplastic tumor type, which portends relatively sticky tissue and cells. Moreover, as pancreatic tumor specimens available for research also tend to be relatively small, efforts are made to maximize the quantity of cells captured.
Isolation of nuclei requires the most optimization in terms of cell lysis conditions and timing, as well as reagent types and ratios. Handling the nuclei over the course of isolation also requires great care. In this article, we describe our experience optimizing nuclear isolation for the 10x Genomics multiome sequencing platform from solid tumor tissue (Figure 1). We provide recommendations for tissue digestion, cryopreservation of single-cell suspensions (if desired), and nuclear isolation.

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

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

As our knowledge of tumor biology grows and interest to understand the intricacies of how cells interact within the tumor and influence patient outcomes, the importance of analyzing heterogeneous cells across the tumor microenvironment has also increased. Multiome sequencing, which permits the acquisition of single cell RNA and attack sequencing from the same cell in a paired cell fashion provides a significant advance towards this end. However, the quality of the data obtained from these experiments is highly dependent on the quality of the experimental conditions and inputted biological material.This protocol provides a reliable and optimized approach to the isolation of nuclei from solid tumor specimens for multiome sequencing using the 10x Genomics platform. This protocol is reliable using either fresh or frozen single cell suspensions. We provide recommendations for tissue dissociation conditions, cryopreservation of single cell suspensions, and assessment of isolated nuclei.In this protocol, we used human pancreatic ductal adenocarcinoma specimens, which are a primary tissue type of focus in our laboratory. Pancreas cancer represents a highly desmoplastic tumor type, which portends relatively sticky tissue as well as cells. Moreover, as pancreatic tumor specimens available for research also tend to be relatively small, efforts are made to maximize the quantity of the cells captured.

为了从患者实体瘤标本中分离出高质量的细胞核进行多组测序（图1），将肿瘤组织解离并冷冻保存单细胞悬浮液（图2A-D）。然后在计划的多组组捕获时解冻细胞悬液。使用优化的裂解缓冲液试剂和定时进行细胞核捕获，以最大限度地提高质量和产量（图3A-D）。具有代表性的细?...

Watch this Scientific Journal Video about Author Spotlight: Enhancing Nuclei Isolation for Multiome Sequencing in Challenging Tumor Microenvironments at JoVE.com

该方案提供了一种可靠且优化的方法，可使用 10x Genomics 平台从实体瘤标本中分离细胞核进行多组测序，包括组织解离条件、单细胞悬液的冷冻保存和分离细胞核的评估建议。

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

随着我们对肿瘤生物学知识的不断加深，以及人们对了解肿瘤内细胞如何相互作用并影响患者预后复杂性的兴趣，分析肿瘤微环境中异质细胞的重要性也有所增加。多组测序允许以配对细胞方式从同一细胞获取单细胞 RNA 和攻击测序，为此取得了重大进展。然而，从这些实验中获得的数据的质量在很大程度上取决于实验条件和输入的生物材料的质量。该协议提供了一种可靠且优化的方法，可使用 10x Genomics 平台从实体瘤标本中分离细胞核以进行多组测序。该方案使用新鲜或冷冻的单细胞悬浮液是可靠的。我们为组织解离条件、单细胞悬液的冷冻保存和分离细胞核的评估提供建议。在该方案中，我们使用了人胰腺导管腺癌标本，这是我们实验室的主要组织类型。胰腺癌是一种高度纤维增生的肿瘤类型，预示着相对粘性的组织和细胞。此外，由于可用于研究的胰腺肿瘤标本也往往相对较小，因此努力最大限度地提高捕获的细胞数量。

optimized-nuclei-isolation-from-fresh-frozen-solid-tumor-specimens

Research

JoVE Journal

Cancer Research

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

786 次观看.  Stanford University. 随着我们对肿瘤生物学知识的不断加深，以及人们对了解肿瘤内细胞如何相互作用并影响患者预后复杂性的兴趣，分析肿瘤微环境中异质细胞的重要性也有所增加。多组测序允许以配对细胞方式从同一细胞获取单细胞 RNA 和攻击测序，为此取得了重大进展。然而，从这些实验中获得的数据的质量在很大程度上取决于实验条件和输入的生物材料的质量。该协议提供了一种?...