作者感谢 Filip Bochner、Marion Richardson、Petra Staeuble 和 Matthias Selhausen 提供本手稿中分析的动物组织。我们还要感谢Petra Schwalie、Klas Hatje、Roland Schmucki和Martin Ebeling对生物信息学的支持。

所有动物研究均在严格遵守瑞士联邦动物保护法规的情况下，经巴塞尔城市州兽医局批准，或经机构动物护理和使用委员会批准，符合《德国动物福利法》。
1. 组织和试剂/仪器制备
<ol><li>清洁和仪器准备<ol><li>用 70% 乙醇和 RNase 去污溶液清洁工作台和镊子。将离心机预冷至4°C。</li>
		<li>将细胞核分离柱在4°C的冰箱中预冷至少30分钟。</li>
		<li>启动机器人解离器并打开冷却，方法是将屏幕右上角的滑块设置为冷却，然后单击它开始冷却，使滑块显示为橙色。检查连接的细胞核储存试剂 （NSR） 瓶和细胞核分离缓冲液 （NIB） 瓶是否有足够的液体剩余并正确冷却。</li>
		<li>准备一个装满干冰的聚苯乙烯泡沫盒，并在干冰上预冷培养皿和手术刀刀片。</li>
	</ol></li>
	<li>缓冲液制备<ol><li>制备1.5M蔗糖缓冲溶液（SCS），如 表1所示。将 SCS 分装到 2 mL DNase/RNase 管中的 500 μL 等分试样中，每个样品获得 4 份 500 μL SCS 等分试样。将等分试样放在冰上，直到进一步使用。</li>
		<li>在处理脑组织的情况下，通过将二氧化硅胶体储备溶液稀释在NSR中并加入RNase抑制剂，制备 表2中所述的18%二氧化硅胶体溶液。每个样品制备 3 mL 18% 二氧化硅胶体溶液，并将其放在冰上。</li>
	</ol></li>
</ol>2. 组织匀浆和细胞核分离
<ol><li>从-80°C冰箱中取出样品，并立即将其置于干冰上。</li>
	<li>用预冷的手术刀将样品在预冷的培养皿或干冰上的金属板上切成15-50mg的碎片（如果尺寸还不合适）。确保以正确的方向切割样品，以便样品仍能代表感兴趣的器官结构。 注意：使用此方案，15-50mg是核提取的最佳样本量。为了在净化后获得良好的产量，建议样品量至少为 25 mg。对于较小的样品，可以使用特殊的滤芯，这些滤芯经过优化，可使用机器人解离器处理少量输入。采用小起始细胞核分离柱解离重量低至 4 mg 的组织样品，为下游单核 RNA 测序提供足够的产量。使用来自大鼠肝组织的低起始柱的细胞核产量示例如 表3所示。</li>
	<li>从冰箱中取出细胞核分离盒，打开包装，取出研磨机，然后将 15 μL RNase 抑制剂 （40 U/μL） 移液到柱子底部。 注意：在细胞核提取过程中，机器人解离器会将 NIB 和 NSR 添加到小柱中，总体积为 3 mL（使用低体积细胞核提取方案）。通过在提取前向小柱中加入 15 μL RNase 抑制剂 （40 U/μL），悬浮液将具有所需的 RNAse 抑制剂浓度为 0.2 U/μL。</li>
	<li>使用镊子将组织样本放在墨盒底部。不要将样品完全放置在滤芯的中心，以获得最佳的破坏效率。</li>
	<li>在仪器上选择 运行协议 ，然后单击左上角的 “细胞核 ”选项。<ol><li>从菜单中，选择 低体积核分离 协议，然后单击 “修改”验证中断速度是否设置为快速。打开门并通过抬起红色旋钮滑出载物台，将唱头加载到仪器上。</li>
		<li>将墨盒插入指定位置，旋转墨盒锁，然后滑入载物台，直到红色旋钮卡入到位。关上门，单击 “下一步”开始在仪器上运行细胞核提取。跑步持续约 7 分钟。</li>
	</ol></li>
	<li>运行完成后，提起红色旋钮并拉出载物台，从仪器上取下唱头。立即将墨盒放在冰上。</li>
	<li>对于除大脑以外的所有组织，继续执行步骤 3.1。对于大脑样本，请直接执行步骤 3.2。</li>
</ol>3. 细胞核清理
<ol><li>蔗糖梯度净化 注意：对于脑组织，请跳过此步骤，直接转到步骤 3.2。所有净化步骤均在冰上进行，以尽量减少RNA降解。缓冲液和离心管以及离心机需要预冷。所有重悬和混合步骤仅通过小心移液来执行，因为涡旋可能会损害细胞核的质量和完整性。<ol><li>用移液器吸头小心地刺穿解离器墨盒上的圆箔。 注：解离后，所得细胞核悬浮液的体积约为 2 mL。为了便于蔗糖梯度纯化，将细胞核悬浮液分成两份 900 μL 等分试样，从而在纯化过程中使用总体积为 1.8 mL 的细胞核悬浮液。</li>
		<li>从小柱中取出第一个 900 μL 细胞核悬浮液等分试样，并将其添加到先前在 2 mL 试管中制备的 500 μL SCS 等分试样中。通过移液充分混合，直到混合物均匀。</li>
		<li>取出1400μL细胞核悬浮液-SCS混合物，小心地将其分层到新的500μLSCS等分试样上，方法是将试管保持一定角度并逐滴加入混合物，从而形成清晰可见的相分离（见 图1A）。</li>
		<li>小心地关闭试管并将其放回冰上，而不会干扰相分离。</li>
		<li>重复步骤3.1.2-3.1.4，使用第二个900μL悬浮液等分试样和新的SCS等分试样，每个样品有两个2 mL管，具有清晰可见的相分离，用于梯度离心。</li>
		<li>小心地将试管加入预冷的离心机中，并在4°C下以13,000× g 旋转15分钟。</li>
		<li>同时，通过将RNase抑制剂添加到NSR的等分试样中来制备 表4中描述的NSR。每个样品制备 1 mL NSR。 注：此时，可以从-80°C冰箱中取出单细胞基因表达试剂凝胶珠，使其平衡至室温（RT），并且模板开关寡核苷酸可以重悬于低TE缓冲液中。</li>
		<li>离心后，在不干扰沉淀的情况下从两个试管中取出上清液，并按照制造商的建议小心地将沉淀重悬于 50 μL 冰冷的 NSR 中。将同一样品的两个沉淀汇集到一个新的 1.5 mL 管中，并加入 900 μL 冰冷的 NSR，总体积为 1 mL。通过移液充分混合。</li>
		<li>用摆动桶转子离心机在4°C下以500× g 离心样品5分钟。 注意：强烈建议使用水平转子来最大限度地减少细胞核损失，特别是当细胞核产量预计较低或开始时需要少量组织时。</li>
		<li>同时，如 表5所述，用0.04%牛血清白蛋白（BSA）和0.2U/μL RNase抑制剂制备每个1x PBS（不含Ca2 +和Mg2+）样品500μL。</li>
		<li>小心地除去上清液，不要丢弃沉淀，并将沉淀重悬于100μLPBS溶液中，如上所述（1x PBS + 0.04%BSA + RNase抑制剂，0.2U / μL）。 注：对于小组织样品，细胞核浓度可能较低，因此，建议仅将沉淀重悬于50μL的PBS溶液中，以确保单核RNA测序的浓度足够高。</li>
		<li>直接继续执行步骤 4。</li>
	</ol></li>
	<li>二氧化硅胶体梯度净化 注意：对于脑组织，二氧化硅胶体梯度比蔗糖梯度更适合去除细胞核悬浮液中的髓鞘和碎屑。所有净化步骤均在冰上进行，以尽量减少RNA降解。缓冲液和离心管以及离心机都需要预冷。所有重悬和混合步骤仅通过小心移液来执行，因为涡旋可能会损害细胞核的质量和完整性。<ol><li>用移液器吸头小心地刺穿解离器墨盒上的圆箔。</li>
		<li>从试剂盒中取出细胞核悬浮液，并将其加入 5 mL 试管中。</li>
		<li>在预冷的离心机中以500× g 在4°C离心5分钟。</li>
		<li>小心地除去上清液，不要干扰沉淀，并将沉淀重悬于1mL冰冷的18%二氧化硅胶体溶液中。</li>
		<li>再加入 2 mL 的 18% 二氧化硅胶体溶液，使总体积为 3 mL，并通过移液充分混合。</li>
		<li>将样品在4°C下以700× g 离心5分钟，在摆动桶转子中，制动器关闭。</li>
		<li>同时，通过将RNase抑制剂加入NSR的等分试样中来制备 表4 中描述的NSR。每个样品制备 1 mL NSR。 注：此时，可以从-80°C冰箱中取出单细胞基因表达试剂凝胶珠，使其平衡至RT，模板开关寡核苷酸可以重悬于低TE缓冲液中。</li>
		<li>小心地从离心机中取出样品，不要干扰漂浮在顶部的髓鞘层。</li>
		<li>首先，从顶部去除髓鞘层并丢弃;然后，小心地除去整个上清液，不要干扰沉淀。 注意：通过将无菌无绒擦拭布包裹在 1 mL 移液器吸头上以吸出髓鞘层和 1-2 mL 上清液，可以轻松去除髓鞘层。</li>
		<li>根据制造商的建议，将沉淀重悬于 1 mL 冰冷的 NSR 中。</li>
		<li>在带有摆动桶转子的离心机中，在4°C下以500× g 离心样品5分钟。 注意：强烈建议使用水平转子来最大限度地减少细胞核损失，特别是当细胞核产量预计较低或开始时需要少量组织时。</li>
		<li>同时，如 表5所述，用0.04%牛血清白蛋白（BSA）和0.2U/μL RNase抑制剂制备每个1x PBS（不含Ca2 +和Mg2+）样品500μL。</li>
		<li>小心地除去上清液，不要干扰沉淀，并将样品重悬于100μL如上制备的PBS溶液中（1x PBS + 0.04%BSA + RNase抑制剂，浓度为0.2U / μL）。 注：对于小组织样品，细胞核浓度可能较低，因此，建议仅将沉淀重悬于50μL的PBS溶液中，以确保单核RNA测序的浓度足够高。</li>
	</ol></li>
</ol>4. 计数
<ol><li>对于每个待计数的样品，在 20 μL PBS 溶液中稀释 10 μL 细胞核悬浮液以获得 1：3 的稀释度。</li>
	<li>为了计数，将 25 μL 碘化丙啶 （PI） 染色溶液加入荧光计数板的混合孔中。加入 25 μL 稀释的细胞核悬浮液，并通过移液充分混合。将 50 μL 染色样品从混合孔转移到上样孔中。</li>
	<li>将计数板加载到细胞计数器上并开始计数。 注意：细胞核计数取自红色荧光通道，曝光时间为 700 ms。该通道经过优化，通过与在显微镜下使用Neubauer Chamber和台盼蓝染色的手动计数进行交叉比较，获得准确的细胞核计数。在高浓度下，原子核非常靠近，使得软件难以将它们分开。在这种情况下，建议在合适的稀释度中重新计数样品。原子核的完整性和清洁度可以通过明场图像或显微镜下进行评估。</li>
	<li>用PBS（1x PBS + 0.04%BSA + RNase抑制剂，浓度为0.2U / μL）稀释样品至单核RNA测序所需的浓度。 注：浓度在 700-1,200 个细胞核/μL 之间被认为是单核 RNA 测序的最佳选择。较低的细胞浓度（例如 700 个细胞核/μL）可减少环境 RNA 的背景污染。</li>
</ol>5. 文库准备
<ol><li>使用制造商的方案使用单细胞基因表达试剂进行单核RNA测序，目标是每个样品的细胞核回收率为8000-10,000个细胞核。</li>
</ol>6. 测序
<ol><li>使用双端、双索引对具有所需测序深度的文库进行测序，测序读数如下：读数 1：28 个循环，i7 索引：10 个循环，i5 索引：10 个循环和读数 2：90 个循环。</li>
</ol>

从冷冻哺乳动物组织中分离和分析单个细胞核

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B., 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+comparative+strategy+for+single-nucleus+and+single-cell+transcriptomes+confirms+accuracy+in+predicted+cell-type+expression+from+nuclear+RNA.">A comparative strategy for single-nucleus and single-cell transcriptomes confirms accuracy in predicted cell-type expression from nuclear RNA.</a> Scientific Reports. 7, 6031 (2017).</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> Journal of Visualized Experiments: JoVE. (162), 61542 (2020).</li><li>Jovanovich, 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=.">.</a> Automated processing of solid tissues into single cells or nuclei for genomics and cell biology applications with the Singulator&#8482; 100 and 200 systems. , (2022).</li><li>Bell, 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=Characterization+of+a+novel+high-throughput,+high-speed+and+high-precision+plate-based+image+cytometric+cell+counting+method.">Characterization of a novel high-throughput, high-speed and high-precision plate-based image cytometric cell counting method.</a> Cell &amp; Gene Therapy Insights. 7 (4), 427-447 (2021).</li><li>Madler, S. C., 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=Besca,+a+single-cell+transcriptomics+analysis+toolkit+to+accelerate+translational+research.">Besca, a single-cell transcriptomics analysis toolkit to accelerate translational research.</a> NAR Genomics and Bioinformatics. 3 (4), lqab102 (2021).</li><li>Wu, H., 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=Mapping+the+single-cell+transcriptomic+response+of+murine+diabetic+kidney+disease+to+therapies.">Mapping the single-cell transcriptomic response of murine diabetic kidney disease to therapies.</a> Cell Metabolism. 34 (7), 1064-1078 (2022).</li><li>Han, L., 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=Cell+transcriptomic+atlas+of+the+non-human+primate+Macaca+fascicularis.">Cell transcriptomic atlas of the non-human primate Macaca fascicularis.</a> Nature. 604 (7907), 723-731 (2022).</li><li>Madissoon, 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=scRNA-seq+assessment+of+the+human+lung,+spleen,+and+esophagus+tissue+stability+after+cold+preservation.">scRNA-seq assessment of the human lung, spleen, and esophagus tissue stability after cold preservation.</a> Genome Biology. 21 (1), 1 (2019).</li><li>Caglayan, E., Liu, Y., Konopka, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+ambient+RNA+contamination+causes+misinterpreted+and+masked+cell+types+in+brain+single-nuclei+datasets.">Neuronal ambient RNA contamination causes misinterpreted and masked cell types in brain single-nuclei datasets.</a> Neuron. 110 (24), 4043-4056 (2022).</li><li>Luecken, M. D., Theis, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+best+practices+in+single-cell+RNA-seq+analysis:+a+tutorial.">Current best practices in single-cell RNA-seq analysis: a tutorial.</a> Molecular Systems Biology. 15 (6), e8746 (2019).</li><li>Fleming, S. 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=Unsupervised+removal+of+systematic+background+noise+from+droplet-based+single-cell+experiments+using+CellBender.">Unsupervised removal of systematic background noise from droplet-based single-cell experiments using CellBender.</a> bioRxiv. , (2022).</li><li>Yang, 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=Decontamination+of+ambient+RNA+in+single-cell+RNA-seq+with+DecontX.">Decontamination of ambient RNA in single-cell RNA-seq with DecontX.</a> Genome Biology. 21 (1), 57 (2020).</li><li>Young, M. D., Behjati, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SoupX+removes+ambient+RNA+contamination+from+droplet-based+single-cell+RNA+sequencing+data.">SoupX removes ambient RNA contamination from droplet-based single-cell RNA sequencing data.</a> Gigascience. 9 (12), giaa151 (2020).</li></ol>

在研究进行期间，所有作者都是 F. Hoffmann-La Roche 的雇员。

我们开发了一种多功能且部分自动化的方案，用于从冷冻哺乳动物组织获得高质量的单核，并在小鼠脑、大鼠肾脏和食蟹猴肝脏和脾脏组织中展示了该方案。
当将该协议的性能与其他已发表的在脑，肾脏，脾脏和肝组织中进行单核RNA测序的方案进行比较时，我们观察到我们能够检测到相似数量的基因和每个细胞核的UMI计数，并且能够恢复预期的细胞类型。与现有方法相比，该协议有几个优点。首先，本研究中的方案可自动进行组织匀浆和单核分离。这是通过使用机器人组织破坏器21实现的。在大多数方案中，用 Dounce 匀浆器匀浆组织以释放单核 3,20。然而，我们注意到，这个手动步骤会导致原子核产量和完整性的实验变化，这取决于均质化过程中施加的力的大小，从而影响实验的可重复性。在这里，通过使用具有固定设置的自动组织研磨机，在实验中获得了良好的细胞核质量和产量以及更高的一致性。此外，自动化此步骤还减少了方案的手动操作时间（组织破碎步骤大约需要 7 分钟），使用户能够为后续步骤做好准备。其次，本研究中描述的方案是通用的，即它与来自不同物种的不同组织兼容。这使我们能够避免冗长的方案优化，例如，识别不同组织的均质缓冲液/去污剂2,5,6。第三，该协议不依赖于访问流动分选机以获得干净的细胞核，因此对于没有流动分选所需设备/专业知识的实验室来说，该协议更容易获得。相反，我们优化了基于蔗糖梯度的过滤，以去除大部分碎屑。然而，特别是对于脑组织，建议使用二氧化硅胶体梯度而不是蔗糖梯度，以更有效地去除髓鞘。我们还发现，在蔗糖/二氧化硅胶体梯度离心步骤结束时使用水平转子可以最大限度地减少细胞核损失。因此，强烈建议使用这种转子。第四，在测试了多种细胞核计数方法（在显微镜下手动计数，使用多个自动计数器）后，建议使用自动荧光细胞计数器22。使用 DNA 嵌入染料（如碘化丙啶）可提高细胞核计数的准确性。第五，该协议从开始到加载微流控芯片大约需要75分钟。这有助于确保在处理多个样品时细胞核完整性保持高水平。最后，我们发现该协议也与最佳切割温度化合物（OCT）包埋的组织兼容。如果使用这种材料，可以在均质化之前使用手术刀将组织从 OCT 块中取出。
单核 RNA 测序数据集中一个常见的挑战是环境 RNA 的存在，它可以是非核的（例如线粒体），也可以是核衍生的27,28。在我们的实验方案中，线粒体RNA（非核环境RNA的代理）即使在过滤之前也很低（所示组织的0.1-1.6%）。然而，与其他方案和数据集类似，来自丰富细胞类型（如肝脏中的肝细胞、大脑中的神经元等）细胞核中高表达基因的环境RNA污染仍然存在27。存在几种生物信息学工具，如 CellBender、SoupX 等，可以在细胞核注释之前去除这种环境 RNA 污染 29,30,31。该协议的另一个局限性是，尽管组织破碎和细胞核分离步骤是自动化的，但该步骤的通量仍然受到限制，因为一次只能处理一个样品。然而，由于此步骤每块组织仅需要大约 7 分钟，因此仍然可以批量处理多个样品。我们通常每批处理四个样品，但每批最多处理六个样品，效果很好。机器人解离器最近进行了改进，允许同时并行处理两个样品，这将使每批处理 8-12 个样品成为可能，这与用于单核封装的微流控芯片的通量兼容。
虽然我们尚未将该协议分离的细胞核用于其他下游应用，例如使用其他平台的ATAC-seq或snRNAseq，但基于此处使用的基因表达试剂获得的数据质量，我们认为我们的方案应该与其他下游应用兼容。然而，未来的工作将涉及使用其他下游应用程序（如ATAC-seq）测试该协议。
总之，我们开发了一种快速、简单且部分自动化的细胞核分离方案，用于下游单核RNA测序，该方案已被证明与不同类型的冷冻哺乳动物组织兼容。

单细胞 （sc） 和单核 （sn） RNA 测序已成为分子和细胞生物学中常用的方案，因为与批量 RNA 测序相比，基因表达的分辨率更高。然而，从实体组织中分离高质量的单细胞和单核制剂仍然是一个挑战，并且通常是sc/sn-RNAseq实验中的限速步骤。事实上，已经开发了大量的方案，使用各种化学和机械程序来获得细胞/细胞核悬浮液 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15 .此外，从碎屑/团块等中清除此类制剂的策略包括流动分选、过滤和洗涤。此类方案通常是手动的（导致与用户相关的变异性），可能很耗时（导致细胞/细胞核活力降低），和/或可能需要使用流式细胞仪进行细胞/细胞核分选。本研究的重点是开发一种简单、快速且部分自动化的从冷冻哺乳动物组织中分离单核方案，用于下游 RNA 测序应用。我们特别关注细胞核分离，而不是细胞分离，因为它与冷冻组织的使用兼容，使样品收集/处理更加实用，并实现样品的无偏批次，特别是在时间过程实验中。此外，尽管核转录组不能完全反映细胞转录组，但现在已经有几项研究表明，单细胞核RNA测序数据与单细胞RNA测序数据相当，用于细胞类型鉴定，即使细胞类型的比例可以变化6,16,17,18,19。
细胞核分离包括几个步骤：1）对组织进行机械或化学破坏以释放细胞核，2）清理碎片和团块，以及3）准确计数细胞核，为下游应用做准备。在许多方案中，步骤1通常涉及使用Dounce匀浆器以破坏组织3,20。或者，可以使用化学方法，尽管这些方法通常需要针对不同的组织进行优化2,5,6。我们经验到，手动组织破坏程序容易出现与操作者相关的变异性，导致细胞核的质量和产量参差不齐。为了最大限度地减少技术变异性并具有跨组织工作的更一致和可重复的方案，开发了一种使用市售机器人组织解离器的方案21。对于第 2 步，虽然缓冲液置换通常是洗涤细胞核的最简单方法，但我们采用了相对较短的蔗糖梯度离心步骤，以更彻底地去除碎屑。具体到脑组织，我们使用二氧化硅胶体梯度代替蔗糖梯度，以更有效地去除髓鞘。最后，对于计数，使用血细胞计数器是计数和目视检查细胞核的金标准。在我们的实验方案中，可以使用市售的自动荧光细胞计数器22可靠地自动化该步骤。该方案已经过测试，与来自不同哺乳动物物种（大鼠、小鼠和非人灵长类动物）的几种冷冻哺乳动物组织（包括脑、肾脏、脾脏和肝脏）兼容，并通过基于液滴的商业平台为下游单核 RNA 测序提供高质量的细胞核。从组织制备到单核RNA测序工作流程的开始，该方案大约需要75分钟。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 M DTT</td>
			<td>Thermo Fisher Scientific</td>
			<td>P2325</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Tween 20</td>
			<td>Bio-Rad</td>
			<td>1662404</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Magnetic Separator</td>
			<td>10x genomics</td>
			<td>PN-120250</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Vortex Adapter</td>
			<td>10x genomics</td>
			<td>PN-120251</td>
			<td></td>
		</tr>
		<tr>
			<td>1x DPBS (10x), no calcium, no magnesium</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190144</td>
			<td>stored at 4&#176;C</td>
		</tr>
		<tr>
			<td>30% Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A9576_50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>400 mM Tris-HCl, pH 8.0</td>
			<td>Thermo Fisher Scientific</td>
			<td>15568025</td>
			<td></td>
		</tr>
		<tr>
			<td>40U/&#956;l RNaseOUT Recombinant Ribonuclease Inhibitor</td>
			<td>Thermo Fisher Scientific</td>
			<td>10777019</td>
			<td>Stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Agilent High Sensitivity DNA Kit</td>
			<td>Agilent</td>
			<td>5067-4626</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellaca MX High-throughput Automated Cell Counter</td>
			<td>Nexcelom Bioscience</td>
			<td>CELMXSYSF2</td>
			<td>Automated fluorescent cell counter</td>
		</tr>
		<tr>
			<td>Chromium Next GEM Chip G Single Cell Kit, 16 rxns</td>
			<td>10x genomics</td>
			<td>PN-1000127</td>
			<td>Single cell gene expression reagent, stored at room temperature</td>
		</tr>
		<tr>
			<td>Chromium Next GEM Secondary Holder</td>
			<td>10x genomics</td>
			<td>PN-1000195</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromium Next GEM Single Cell 3&#39; Gel Bead Kit v3.1, 4 rxns</td>
			<td>10x genomics</td>
			<td>PN-1000129</td>
			<td>Single cell gene expression reagent, stored at -80 &#176;C</td>
		</tr>
		<tr>
			<td>Chromium Next GEM Single Cell 3&#39; GEM, Library &#38; Gel Bead Kit v3.1, 4 rxns</td>
			<td>10x genomics</td>
			<td>PN-1000128</td>
			<td>Single cell gene expression reagent</td>
		</tr>
		<tr>
			<td>Chromium Next GEM Single Cell 3&#39; Library Kit v3.1 4 rxns</td>
			<td>10x genomics</td>
			<td>PN-1000158</td>
			<td>Single cell gene expression reagent, stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Chromium Next GEM Single Cell 3&#39;GEM Kit v3.1 4 rxns</td>
			<td>10x genomics</td>
			<td>PN-1000130</td>
			<td>Single cell gene expression reagent, stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Divided Polystyrene Reservoirs</td>
			<td>VWR</td>
			<td>41428-958</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA LoBind Tubes 1.5ml Eppendorf</td>
			<td>Sigma-Aldrich</td>
			<td>EP0030108051</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA LoBind Tubes 2ml Eppendorf</td>
			<td>Sigma-Aldrich</td>
			<td>EP0030108078</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry ice</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads MyOne SILANE</td>
			<td>10x genomics</td>
			<td>PN-2000048</td>
			<td>Single cell gene expression reagent, stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Ethanol Pure</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerin (Glycerol), 50% (v/v)</td>
			<td>Ricca Chemical Company</td>
			<td>3290-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Heatblock</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High-Throughput Nexcelom Counting Plates</td>
			<td>Nexcelom Bioscience</td>
			<td>CHM24-A100-001</td>
			<td>Cell counter counting plate</td>
		</tr>
		<tr>
			<td>Low TE Buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12090015</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Centrifuge</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>NovaSeq 6000 SP Reagent Kit v1.5 (100 cycles)</td>
			<td>Illumina</td>
			<td>2002840</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclei Isolation Buffer</td>
			<td>S2 Genomics</td>
			<td>100-063-396</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Nuclei Isolation Cartridge</td>
			<td>S2 Genomics</td>
			<td>100-063-287</td>
			<td>Precooled at 4 &#176;C before use</td>
		</tr>
		<tr>
			<td>Nuclei PURE 2 M Sucrose Cushion Solution</td>
			<td>Sigma-Aldrich</td>
			<td>NUC201-1KT</td>
			<td>Sucrose cushion solution&#160;</td>
		</tr>
		<tr>
			<td>Nuclei PURE Sucrose Cushion Buffer</td>
			<td>Sigma-Aldrich</td>
			<td>NUC201-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclei Storage Reagent</td>
			<td>S2 Genomics</td>
			<td>100-063-405</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>PCR Tubes 0.2 ml 8-tube strips</td>
			<td>Eppendorf</td>
			<td>30124359</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE Healthcare</td>
			<td>17-0891-02</td>
			<td>Silica colloid solution</td>
		</tr>
		<tr>
			<td>PhiX Control v3</td>
			<td>Illumina</td>
			<td>FC-110-3001</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen Buffer EB</td>
			<td>Qiagen</td>
			<td>19086</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>Q32854</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated Centrifuge (Eppendorf 5804R)</td>
			<td>Eppendorf&#160;</td>
			<td>5805000010</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated Centrifuge with Swinging-Bucket Rotor (Eppendorf 5810R)</td>
			<td>Eppendorf&#160;</td>
			<td>5811000015</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAseZap</td>
			<td>Ambion</td>
			<td>AM9780</td>
			<td>RNAse decontamination solution</td>
		</tr>
		<tr>
			<td>Round cell culture petri dish</td>
			<td>SPL</td>
			<td>330005</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel disposable</td>
			<td>Aesculap AG</td>
			<td>BA210</td>
			<td>pre-cooled on dry ice before use</td>
		</tr>
		<tr>
			<td>Single Index Kit T Set A, 96 rxns</td>
			<td>10x genomics</td>
			<td>PN-1000213</td>
			<td>Single cell gene expression reagent, stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Singulator 100 System</td>
			<td>S2 Genomics</td>
			<td>-</td>
			<td>Commercially available robotic tissue dissociator</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide 1M</td>
			<td>Sigma-Aldrich</td>
			<td>72068</td>
			<td></td>
		</tr>
		<tr>
			<td>SPRIselect Reagent Kit</td>
			<td>Beckman Coulter</td>
			<td>b23318</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile tweezers</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure DNase/RNase-Free Distilled Water</td>
			<td>Thermo Fisher Scientific</td>
			<td>10977049</td>
			<td></td>
		</tr>
		<tr>
			<td>ViaStain PI Staining Solution</td>
			<td>Nexcelom Bioscience</td>
			<td>CS1-0109-5mL</td>
			<td>Propidium iodide staining solution</td>
		</tr>
		<tr>
			<td>Vortex Mixer+A2:D44</td>
			<td>VWR</td>
			<td>-</td>
			<td></td>
		</tr>
	</tbody>
</table>

a simple, quick, and partially automated protocol for the isolation of single nuclei from frozen mammalian tissues for single nucleus sequencing

单细胞和单核RNA测序由于提供了丰富的转录组学信息，已成为常见的实验室应用。特别是单核RNA测序，可用于研究难以解离组织中的基因表达。此外，这种方法还与冷冻（存档）材料兼容。在这里，我们描述了一种从冷冻哺乳动物组织中分离高质量单核的方案，用于使用市售仪器和试剂以部分自动化方式进行下游单核RNA测序。具体来说，机器人解离器用于自动化和标准化组织均质化，然后优化化学梯度过滤细胞核。最后，我们使用自动荧光细胞计数仪准确、自动地对细胞核进行计数。该方案的性能在小鼠脑，大鼠肾脏和食蟹猴肝脏和脾脏组织上得到证明。该方案简单、快速且易于适应各种哺乳动物组织，无需大量优化，并为下游单核 RNA 测序提供高质量的细胞核。

通过对来自三只B6小鼠的新鲜冷冻脑枕叶皮层组织，来自三只Wistar大鼠的新鲜冷冻横向切割肾组织，来自三只毛里求斯食蟹猴的存档（11岁）肝脏和脾脏组织进行单核RNA测序，证明了该协议的性能和多功能性。所有动物均未灌注。
如图1B，C所示，获得了没有起泡、碎屑和结块迹象的高质量原子核。基于蔗糖梯度的过滤经过优化，通过测试不同的密度、离心速度和时间，在显微镜下评估细胞核纯度/完整性，以及评估细胞核尺寸分布和产量，去除大部分碎片（图1D）。这使我们能够选择1.5 M的蔗糖梯度密度，并使用15 min的短离心时间。接下来，为了进一步评估细胞核的质量，使用10X Cell Ranger对数据进行预处理，并使用Besca23进行进一步的下游数据分析。过滤掉线粒体含量>5%的细胞核（因为这些细胞核往往是受损/应激的细胞核），并保留具有500-7,000个基因的细胞核（以尽量减少空液滴和多重）。我们只纳入了至少30个细胞核中存在的基因。我们针对每个大脑皮层样本 8,000 个细胞核，每个肾脏、肝脏和脾脏样本 10,000 个细胞核。经过滤，从3个脑样本中获得了10,644个高质量细胞核，从3个肾脏样本中获得了14,960个高质量细胞核，从3个肝脏样本中获得了18,795个高质量细胞核，从3个脾样本中获得了13,882个高质量细胞核。图 2A、D、G、J 显示了代表每个样品中 UMI 计数、基因计数和线粒体含量分布的小提琴图。所有脑样本的计数中位数为 7,563 个 UMI/细胞核和 3,208 个基因/细胞核。所有肾脏样本的计数中位数为 3,841 个 UMI/细胞核和 1,915 个基因/细胞核。所有肝脏样本的计数中位数为 2,649 个 UMI/细胞核和 1,676 个基因/细胞核。所有脾脏样本的计数中位数为 1,609 个 UMI/细胞核和 1,138 个基因/细胞核。然后，我们使用高度可变的基因生成簇，并使用已知的标记基因17,24,25,26对其进行注释。如图2B，E，H，K所示，我们能够从每个组织中识别出预期的细胞类型。此外，如图2B，E，H，K所示，所有动物都对所有集群做出了贡献，表明该协议引入的整体技术变异性较低。此外，每种组织类型的所有三个样品的细胞比例具有可比性，UMI和基因计数也是如此（图2A，C，D，F，G，I，J，L）。一个值得注意的例外是肝脏，其中三个肝脏样本中的肝细胞群在比例和轮廓上有所不同。这很可能是由于动物之间的生物学差异（性别、年龄、代谢状态）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65611/65611fig01.jpg"> 图 1：细胞核质量和蔗糖梯度优化的评估 。 （A） 蔗糖梯度离心过程中的预期相分离用箭头表示。（B）用方案获得的碘化丙啶染色的大鼠肾脏（上）和食蟹猴脾（下）细胞核的代表性荧光图像。（C）从小鼠肝脏（顶部）和小鼠大脑（底部）分离的细胞核的代表性明场显微镜图像，比例尺为500μm。注意原子核的规则光滑表面，表明良好的核质量。（D）蔗糖梯度优化。测试了几种蔗糖密度、旋转速度和旋转时间。显示了每种条件下的细胞核、细胞核大小分布和细胞核产量的明场显微镜图像。 <a href="https://www.jove.com/files/ftp_upload/65611/65611fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65611/65611fig02.jpg"> 图2：snRNAseq在小鼠脑枕叶皮层、大鼠肾脏（皮质和髓质）以及食蟹猴肝脏和脾脏上的代表性数据 。 （A） 小提琴图显示每个脑样本的基因/细胞核、UMI/细胞核和线粒体含量百分比的分布。（B）左图：UMAP图显示了每个样本对大脑中识别的簇的贡献。右图：UMAP显示了基于脑组织中标记基因注释的簇的身份。（C） 在 3 个大脑样本中观察到的细胞比例。（D） 小提琴图显示每个肾脏样本的基因/细胞核、UMI/细胞核和线粒体含量百分比的分布。（E） 左图：UMAP 图显示了每个样本对肾脏中鉴定的簇的贡献。右图：UMAP显示了基于肾脏组织中标记基因注释的簇的身份。（F） 在 3 个肾脏样本中观察到的细胞比例。（G） 小提琴图显示每个肝脏样本的基因/细胞核、UMI/细胞核和线粒体含量百分比的分布。（H）左图：UMAP图显示了每个样本对肝脏中鉴定的簇的贡献。右图：UMAP显示了基于肝组织中标记基因注释的簇的身份。（I） 在 3 个肝脏样本中观察到的细胞比例。（J） 小提琴图显示每个脾脏样本的基因/细胞核、UMI/细胞核和线粒体含量百分比的分布。（K） 左图：UMAP 图显示了每个样本对脾脏中鉴定的簇的贡献。右图：UMAP显示了基于脾脏组织中标记基因注释的簇的身份。（L） 在 3 个脾脏样本中观察到的细胞比例。 <a href="https://www.jove.com/files/ftp_upload/65611/65611fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>组件</td>
			<td>库存集中度</td>
			<td>每个样品的体积</td>
			<td>最终浓度</td>
		</tr><tr><td>蔗糖缓冲液</td>
			<td>1 .</td>
			<td>1500微升</td>
			<td>1.5 米</td>
		</tr><tr><td>蔗糖缓冲液</td>
			<td>-</td>
			<td>500微升</td>
			<td>-</td>
		</tr><tr><td>二硫苏糖醇 （DTT）</td>
			<td>1 米</td>
			<td>2微升</td>
			<td>1 毫米</td>
		</tr><tr><td>RNAse 抑制剂</td>
			<td>40 U/微升</td>
			<td>10微升</td>
			<td>0.2 U/微升</td>
		</tr></tbody></table>表1：1.5 M蔗糖缓冲溶液（SCS）的制备。 该溶液用于步骤3.1中净化期间的蔗糖梯度离心，并且每次在开始方案之前都应新鲜制备。在协议期间，始终将 SCS 保持在冰上。此表中提到的解决方案在 材料表中引用。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>组件</td>
			<td>库存集中度</td>
			<td>每个样品的体积</td>
			<td>最终浓度</td>
		</tr><tr><td>二氧化硅胶体储备液</td>
			<td>90%</td>
			<td>600微升</td>
			<td>18%</td>
		</tr><tr><td>细胞核储存试剂 （S2 Genomics）</td>
			<td>-</td>
			<td>2400微升</td>
			<td>-</td>
		</tr><tr><td>RNAse 抑制剂</td>
			<td>40 U/微升</td>
			<td>15微升</td>
			<td>0.2 U/微升</td>
		</tr></tbody></table>表2：18%二氧化硅胶体溶液的制备。 该溶液用于步骤3.2中净化期间的二氧化硅胶体梯度离心，并且每次在开始方案之前都应新鲜制备。在实验方案期间，始终将 18% 二氧化硅胶体溶液保持在冰上。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>组织</td>
			<td>样品重量</td>
			<td>弹药筒</td>
			<td>屈服</td>
		</tr><tr><td>大鼠肝脏</td>
			<td>25 毫克</td>
			<td>细胞核分离滤芯</td>
			<td>每毫克组织 65,000 个细胞核</td>
		</tr><tr><td>大鼠肝脏</td>
			<td>4 毫克</td>
			<td>小输入核隔离盒</td>
			<td>每毫克组织 32,000 个细胞核</td>
		</tr></tbody></table>表 3：蔗糖梯度净化后低起始细胞核分离柱与细胞核分离柱的细胞核产量。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>组件</td>
			<td>库存集中度</td>
			<td>每个样品的体积</td>
			<td>最终浓度</td>
		</tr><tr><td>细胞核储存试剂</td>
			<td>-</td>
			<td>1000微升</td>
			<td>-</td>
		</tr><tr><td>RNAse 抑制剂</td>
			<td>40 U/微升</td>
			<td>5微升</td>
			<td>0.2 U/微升</td>
		</tr></tbody></table>表4：细胞核储存试剂（NSR）的制备。 该溶液在步骤 3-5 的细胞核分离期间以及步骤 3.1.8 的净化期间使用。它可以在4°C下储存长达4个月。在纯化步骤6的离心步骤中，用RNase抑制剂制备新鲜的等分试样。此表中提到的解决方案在 材料表中引用。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="4">1x PBS + 0.04%BSA储备溶液</td>
		</tr><tr><td>组件</td>
			<td>库存集中度</td>
			<td>库存量</td>
			<td>最终浓度</td>
		</tr><tr><td>PBS（无 Ca 2+，无 Mg2+）</td>
			<td>1 倍</td>
			<td>30毫升</td>
			<td>-</td>
		</tr><tr><td>牛血清白蛋白 （BSA）</td>
			<td>30%</td>
			<td>40微升</td>
			<td>0.04%</td>
		</tr><tr><td colspan="4">1x PBS + 0.04% BSA + 0.2 U/μL RNAse 抑制剂</td>
		</tr><tr><td>组件</td>
			<td>库存集中度</td>
			<td>每个样品的体积</td>
			<td>最终浓度</td>
		</tr><tr><td>1x PBS + 0.04% BSA 储备溶液</td>
			<td>-</td>
			<td>500微升</td>
			<td>-</td>
		</tr><tr><td>RNAse 抑制剂</td>
			<td>40 U/微升</td>
			<td>2.5微升</td>
			<td>0.2 U/微升</td>
		</tr></tbody></table>表5：PBS + 0.04%BSA的制备。 在步骤3.1.10的净化结束时，并在计数后将细胞核悬浮液稀释至所需的浓度，用于10X单核RNA测序（计数步骤4.4）。储备溶液可在4°C下储存长达1个月。在纯化步骤6的离心步骤中，用RNase抑制剂制备新鲜的等分试样。

Watch this Scientific Journal Video about Enhancing Drug Discovery - Development of Automated, Standardized Protocols for Nuclei Extraction from Frozen Tissues at JoVE.com

作者聚焦：增强药物发现 - 开发从冷冻组织中提取细胞核的自动化标准化方案 

该研究描述了一种简单、快速和部分自动化的方案，用于从冷冻哺乳动物组织中分离高质量细胞核，用于下游单核RNA测序。

一种简单、快速、部分自动化的方案，用于从冷冻哺乳动物组织中分离单细胞核，用于单细胞核测序

Single-cell and single-nucleus RNA sequencing have become common laboratory applications due to the wealth of transcriptomic information that they ...

这项研究的重点是支持使用单核RNA测序的药物发现研究，以研究潜在治疗方法的有效性和安全性。因此，我们正在为来自各种器官的新鲜冷冻组织样本制定标准化的细胞核提取方案。我们的目标是通过减少非生物变异性来自动化和标准化细胞核提取，以获得一致和可靠的结果。其优点是通过使用机器人解离器实现部分自动化，并且工作流程简单而简短，可应用于不同物种的各种组织样本。拥有标准化的协议可以帮助我们回答有关药物生物分布的问题，并探索我们可能观察到的任何安全信号背后的机制。未来，我们将测试我们的协议在其他下游应用（如ATAC-seq或多组学）的适用性。我们还将尝试建立检测单核中病毒表达的转基因的方案。

a-simple-quick-partially-automated-protocol-for-isolation-single

Research

JoVE Journal

Biology

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

3.7K 次观看.  Roche Innovation Centre Basel. 这项研究的重点是支持使用单核RNA测序的药物发现研究，以研究潜在治疗方法的有效性和安全性。因此，我们正在为来自各种器官的新鲜冷冻组织样本制定标准化的细胞核提取方案。我们的目标是通过减少非生物变异性来自动化和标准化细胞核提取，以获得一致和可靠的结果。其优点是通过使用机器人解离器实现部分自动化，并且工作流程简单而简短，可应用于不同物?...

视频: 作者聚焦：增强药物发现 - 开发从冷冻组织中提取细胞核的自动化标准化方案 

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

一个简单的协议提取血细胞从野生毛毛虫

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

科研

生物学

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

标记的细胞核从亲和力为基础的隔离<em&gt;果蝇</em&gt;组织的基因表达分析

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are inactive due to 5' truncations, ~80-100 of these elements remain retrotransposition competent and propagate to different locations throughout the genome via RNA intermediates. While older L1s are believed to target AT rich regions of the genome, the chromosomal targets of newer, more active L1s remain poorly defined. Here we describe fluorescence in situ hybridization (FISH) methodology that can be used to track patterns of L1 insertion and rates of ectopic L1 incorporation at the single nucleus level. In these experiments, fluorescein isothiocyanate/cyanine-3 (FITC/CY3) labeled neomycin probes were employed to track L1 retrotransposition in vitro in HepG2 cells stably expressing ectopic L1. This methodology prevents errors in the estimation of rates of retrotransposition posed by toxicity and account for the occurrence of multiple insertions into a single nucleus.

Here we employ FISH methodology to track LINE-1 retrotransposition at the single nuclei level in chromosome spreads of HepG2 cell lines stably expressing synthetic LINE-1.

在单核级LINE-1逆转录转座子分析

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

胰腺胰岛与小鼠分离的简单高效协议

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

Droplet Barcoding-Based Single Cell Transcriptomics of Adult Mammalian Tissues

The analysis of single cell gene expression across thousands of individual cells within a tissue or microenvironment is a valuable tool for identifying cell composition, discrimination of functional states, and molecular pathways underlying observed tissue functions and animal behaviors. However, the isolation of intact, healthy single cells from adult mammalian tissues for subsequent downstream single cell molecular analysis can be challenging. This protocol describes the general processes and quality control checks necessary to obtain high-quality adult single cell preparations from the nervous system or skin that enabled subsequent unbiased single cell RNA sequencing and analysis. Guidelines for downstream bioinformatic analysis are also provided.

This protocol describes the general processes and quality control checks necessary for preparing healthy adult mammalian single cells for droplet-based, high throughput single cell RNA-Seq preparations. Sequencing parameters, read alignment, and downstream single-cell bioinformatic analysis are also provided.

基于滴瓣的成年哺乳动物组织单细胞转录瘤

The analysis of single cell gene expression across thousands of individual cells within a tissue or microenvironment is a valuable tool for identifying ...

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

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

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.

从成年小鼠肾脏中分离细胞核以进行单核RNA测序

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are ...

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

The liver is a complex and heterogenous tissue responsible for carrying out many critical physiological functions, such as the maintenance of energy homeostasis and the metabolism of xenobiotics, among others. These tasks are performed through tight coordination between hepatic parenchymal and non-parenchymal cells. Additionally, various metabolic activities are confined to specific areas of the hepatic lobule-a phenomenon called liver zonation. Recent advances in single-cell sequencing technologies have empowered researchers to investigate tissue heterogeneity at a single-cell resolution. In many complex tissues, including the liver, harsh enzymatic and/or mechanical dissociation protocols can negatively affect the viability or the quality of the single-cell suspensions needed to comprehensively characterize this organ in health and disease.
This paper describes a robust and reproducible protocol for isolating nuclei from frozen, archived liver tissues. This method yields high-quality nuclei that are compatible with downstream, single-cell omics approaches, including single-nucleus RNA-seq, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), as well as multimodal omics (joint RNA-seq and ATAC-seq). This method has been successfully used for the isolation of nuclei from healthy and diseased human, mouse, and non-human primate frozen liver samples. This approach allows the unbiased isolation of all the major cell types in the liver and, therefore, offers a robust methodology for studying the liver at the single-cell resolution.

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).

从快速冷冻肝组织中分离细胞核用于单细胞多组学

The liver is a complex and heterogenous tissue responsible for carrying out many critical physiological functions, such as the maintenance of energy ...

Single-Cell Suspension Preparation from Nile Tilapia Intestine for Single-Cell Sequencing

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The preparation of high-quality single-cell suspensions is essential for single-cell level studies such as single-cell RNA or genome sequencing. However, there is no ready-to-use protocol for aquaculture fish species, particularly for the intestine of tilapia. The effective dissociation enzymes vary depending on the tissue type. Therefore, optimizing the tissue dissociation protocol by selecting the appropriate enzyme or enzyme combination to obtain enough viable cells with minimum damage is essential. This study illustrates an optimized protocol to prepare a high-quality single-cell suspension from Nile tilapia intestine with an enzyme combination of collagenase/dispase. This combination is highly effective for dissociation with the utilization of bovine serum albumin and DNase to reduce cell aggregation after digestion. The cell output satisfies the requirements for single-cell sequencing, with 90% cell viability and a high cell concentration. This protocol can also be modified to prepare a single-cell suspension from the intestines of other fish species. This research provides an efficient reference protocol and reduces the need for additional trials in the preparation of single-cell suspensions for aquaculture fish species.

Here, we demonstrate the preparation of a high-quality single-cell suspension of tilapia intestine for single-cell sequencing.

尼罗罗非鱼肠单细胞悬液制备用于单细胞测序

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The ...

Isolation and Transcriptome Analysis of Plant Cell Types

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, which is known as cellular heterogeneity. In recent years, single-cell and cell-type isolation combined with next-generation sequencing (NGS) techniques have become important tools for studying biological processes at single-cell resolution. However, isolating plant cells is relatively more difficult due to the presence of plant cell walls, which limits the application of single-cell approaches in plants. This protocol describes a robust procedure for fluorescence-activated cell sorting (FACS)-based single-cell and cell-type isolation with plant cells, which is suitable for downstream multi-omics analysis and other studies. Using Arabidopsis root fluorescent marker lines, we demonstrate how particular cell types, such as xylem-pole pericycle cells, lateral root initial cells, lateral root cap cells, cortex cells, and endodermal cells, are isolated. Furthermore, an effective downstream transcriptome analysis method using Smart-seq2 is also provided. The cell isolation method and transcriptome analysis techniques can be adapted to other cell types and plant species and have broad application potential in plant science.

The feasibility and effectiveness of high-throughput scRNA-seq methods herald a single-cell era in plant research. Presented here is a robust and complete procedure for isolating specific Arabidopsis thaliana root cell types and subsequent transcriptome library construction and analysis.

植物细胞类型的分离和转录组分析

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, ...

用于单核测序或免疫染色的血管纹显微切割

Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining

Endocochlear potential, which is generated by the stria vascularis, is essential to maintain an environment conducive to appropriate hair cell mechanotransduction and ultimately hearing. Pathologies of the stria vascularis can result in a decreased hearing. Dissection of the adult stria vascularis allows for focused single-nucleus capture and subsequent single-nucleus sequencing and immunostaining. These techniques are used to study stria vascularis pathophysiology at the single-cell level. 
Single-nucleus sequencing can be used in the setting of transcriptional analysis of the stria vascularis. Meanwhile, immunostaining continues to be useful in identifying specific populations of cells. Both methods require proper stria vascularis dissection as a prerequisite, which can prove to be technically challenging.

作者聚焦： 用于单核测序或免疫染色的成年小鼠血管纹解剖  

The stria vascularis is vital to the generation of endocochlear potential. Here, we present the dissection of the adult mouse stria vascularis for single-nucleus sequencing or immunostaining.