Name: target cell pre-enrichment and whole genome amplification for single cell downstream characterization
Uploaded: 2018-05-15T12:30:00.000+00:00
Duration: 10 min 12 s
Description: Watch this Scientific Journal Video about Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization at JoVE.com

这项研究由奥地利科学基金 (FWF) 资助, 项目没有。I 1220-B19 (附注) 作为 ERANET 项目的一部分, 在翻译癌症研究 (TRANSCAN) "循环肿瘤细胞作为生物标志物的最小残留疾病的前列腺癌 (反恐委员会扫描)"。博士候选人南卡罗来纳州在格拉茨医科大学博士计划分子医学的框架内接受培训。作者感激地承认尼娜 Schlögl、丹尼尔库默尔、加比 Wendt、克劳迪亚 Chudak、茱莉亚. 舒尔茨和乔安娜. 席勒为他们提供专家技术援助。作者感谢 Peinhaupt 的图形设计支持。

所有程序都已获得格拉茨医科大学道德委员会 (25-240 前 12/13) 的批准。外周血用于扣球实验的样本来自健康的个体。
注: 本协议描述从 PBS 中分离 HT-29 细胞 (人结肠癌细胞系) 或从 HT-29 细胞和外周血的人工混合。同样的实验与两个额外的细胞系 (LNCaP 和 VCaP, 实验数据的代表性结果) 进行, 并可以在理论上执行所有细胞表达 EpCAM。
1. 靶细胞的制备
<ol><li>细胞的细胞培养和标记 注意: 在这个协议中, 细胞是培养在 75 cm²培养瓶。如果使用其他细胞培养设备 (例如25 cm²培养皿、6孔板、等), 请相应地调整试剂的数量。所有液体使用在水浴前预热到37°c。如果不另行说明, 则所有协议步骤均在室温 (RT) 中执行。<ol>
<li>培养 HT-29 细胞在麦考伊的5a 改良培养基补充2毫米 l-谷氨酰胺, 20 毫米 Hepes, 10% 胎牛血清 (血清), 1% 青霉素/链霉素在37摄氏度在 5% CO2大气。</li>
		<li>当细胞90% 汇合时, 取出培养基, 用10毫升的 1x PBS 冲洗细胞。</li>
		<li>要收获细胞, 添加2毫升的细胞脱离溶液 (材料表和试剂) 到细胞和孵化细胞约5分钟在37摄氏度。 注: 分离液中含有蛋白水解和 collagenolytic 酶的 1x PBS, 0.5 毫米乙二胺乙酸 (EDTA), 3 毫克/升苯酚红色。将细胞脱离的预热时间减少到最低限度, 因为这将在1小时后不活跃, 在37摄氏度。覆盖整个细胞层以获得最佳细胞脱离。</li>
		<li>用倒置显微镜检查细胞的脱离。</li>
		<li>将所有分离的细胞转移到50毫升管预先填入与10毫升细胞培养培养基 (相同的步骤1.1.1 中使用) 和并用重悬细胞吹打。</li>
		<li>将剩余的电池悬浮液放在 RT 上的卧式滚筒搅拌机上, 然后进入标签步骤。</li>
	</ol>
</li>
	<li>靶细胞的活细胞标记<ol>
<li>稀释1µL 5 毫米 CFSE (供应在二甲基亚砜, 亚砜) 在1毫升 1x PBS 预热在37摄氏度, 以获得5µM 现成使用 CFSE 标签解决方案。 注: 为了获得最佳染色效果, 请使用新的准备好的解决方案。</li>
		<li>在 1x PBS 中准备一个随时可用的 DNA 染色溶液 (材料和试剂表), 并将其存储在 2-6 摄氏度的保护下免受光照。 注: 为了获得最佳染色效果, 请使用新的准备好的解决方案。</li>
		<li>从水平滚子混合器 (步骤 1.1.6) 中获取单元格, 并将单元格放置在 300 x g上以10分钟. 取出上清, 并并用重悬10毫升 1x PBS 中的细胞。</li>
		<li>再次冲洗细胞与 1x PBS 和并用重悬细胞颗粒在500µL 准备使用 CFSE 标签解决方案。</li>
		<li>在37摄氏度的细胞孵育15分钟, 然后在 300 x g的离心后收集细胞3分钟。</li>
		<li>并用重悬1毫升的预预热细胞培养培养基中的标记细胞, 并允许细胞再生37摄氏度30分钟。</li>
		<li>通过离心 300 x g以3分钟的方式收割细胞, 并在1毫升的准备使用的 DNA 染色溶液中并用重悬细胞颗粒, 在37摄氏度为10分钟。</li>
		<li>颗粒细胞, 去除上清和并用重悬细胞在4毫升 1x PBS。使用 hemocytometer 对细胞密度进行评估, 并使用装有 4 '、6-diamidino-2-苯基吲哚 (DAPI) 和荧光素异硫氰酸酯 (FITC) 过滤器(图2A 和 2E) 的荧光显微镜检查荧光标签. </li>
		<li>离心细胞在 300 x g为3分钟, 并并用重悬细胞颗粒根据所需的细胞密度的各自实验, 如给在下一节和地方在冰上。</li>
	</ol>
</li>
</ol>2. 充电导线
注: 细胞可以从目标细胞/外周血史派金在毫升尺度或提供低数量的细胞在微升的比例。前者的方法允许模仿外周血中的稀有细胞条件, 但后者可能是附加少量细胞以进行协议优化/测试的首选方法。
<ol><li>用大量细胞悬浮剂对导线进行充电<ol>
<li>通过将0.8 克 bsa 分解为40毫升 1x PBS (细胞培养使用), 制备 2% bsa 阻断溶液。在 10-20 分钟的水平滚筒搅拌机上, 通过初始涡流和后续孵化, 将 BSA 溶解。</li>
		<li>用2毫升 2% BSA 冲洗空 EDTA 管/肝素钠管, 除去抗凝剂残留物并丢弃溶液。用5毫升 2% BSA 在水平滚筒搅拌机上以 15 rpm 为30分钟, 通过在 RT 上孵化的方式阻断管表面, 并丢弃溶液。</li>
		<li>稀释标记细胞 (1.2 节) 到密度为 100 000 细胞/毫升, 并使用5毫升来充电导线。</li>
		<li>添加5毫升的标记细胞悬浮 (总共 500 000 细胞) 到管。在添加细胞悬浮液时避免气泡。</li>
		<li>卸下存储舱内的电线, 以及持有电线的橡胶帽, 并在 1x PBS 中冲洗 (图 3)。</li>
		<li>在注射器针 (20G) 的帮助下穿透 EDTA 管的橡胶管帽 (步骤 2.1.4), 插入导线, 使功能部分完全沉浸在细胞悬浮中, 当瓶盖回到管子上, 管放在倾斜的滚筒搅拌机上时。 注: 在整个充电过程中, 导线的三重螺旋功能部分应覆盖电池悬浮。</li>
		<li>将倾斜滚筒搅拌机上的管子旋转5转30分钟, 以允许电池附着在导线上。</li>
		<li>用5毫升 1x pbs 冲洗电线, 在 2-6 摄氏度的15毫升管中将其存储在黑暗中, 直到目视检查。 注意: 电线的功能部分必须始终被淹没在 PBS 中, 以避免损害细胞。</li>
	</ol>
</li>
	<li>将靶细胞与外周血人工混合物隔离 (替代充电方法: 2.1。用大量的细胞悬浮物充电导线)<ol>
<li>稀释标记细胞 (1.2 节) 获得高细胞密度的细胞悬浮 (> 1 000 000 细胞/毫升), 从而保持细胞悬浮量增加到外周血低。</li>
		<li>增加500到 500 000 细胞到5毫升的外周血, 并通过反转管混合。</li>
		<li>将电线从存储舱中取下, 取下持有电线的橡胶帽, 并在 1x PBS 中冲洗。</li>
		<li>使用注射器针 (20G) 穿透新的管帽与设备的非功能部分, 使功能部分完全沉浸在尖血的时候, 当瓶盖是回到管和管放置在倾斜的滚筒搅拌机。 注: 在整个充电过程中, 导线的三重螺旋功能部分应覆盖电池悬浮。</li>
		<li>在5转30分钟的 RT 上, 在倾斜的滚筒搅拌机上孵化管。</li>
		<li>在 1x pbs 中冲洗电线3次, 并将其存储在15毫升的管中, 包含 1x pbs, 直到目视检查。 注意: 电线的功能部分必须始终被淹没, 以避免损害细胞。</li>
	</ol>
</li>
	<li>从低容量电池悬浮线充电 (替代充电方法: 2.1。用大量的细胞悬浮物充电导线)<ol>
<li>在 1x PBS 中并用重悬 1 000 000 细胞/毫升的标记细胞。</li>
		<li>在机架上水平固定150毫米一次性玻璃巴斯德吸管。</li>
		<li>将电线从存储舱中取出, 但要保持黄色橡胶帽持有导线。</li>
		<li>将导线放在吸管中, 使功能性部分位于不接触玻璃的巴斯德吸管的尖端。 注意: 导线的尖端不应伸出巴斯德吸管尖端。避免堵塞巴斯德吸管尖端的后端与持有电线的橡胶帽。如果连接紧, 细胞悬浮不能从另一侧加载到吸管尖端。</li>
		<li>负载15µL 的细胞悬浮到巴斯德吸管的尖端, 覆盖功能性的部分电线。</li>
		<li>每分钟用10分钟的时间, 将组装好的电线/巴斯德吸管尖孵化出来。</li>
		<li>从巴斯德吸管尖端拔下电线, 在5毫升的 1x pbs 中冲洗, 并将其存储在 2-6 °c 的15毫升管中, 包括 1x pbs, 直到目视检查。 注意: 功能部分必须始终被淹没在 1x PBS, 以避免损害细胞。</li>
	</ol>
</li>
</ol>3. 计算导线上的细胞数
注意: 始终保持线的功能部分淹没在 1x PBS, 以避免损害细胞。
<ol><li>使用润滑脂笔在玻璃滑梯上绘制矩形区域, 并添加500µL 1x PBS。</li>
	<li>将导线的非功能部分弯曲, 将其放在玻璃滑梯上, 使功能部分浸入 1x PBS 中。 注: 适合矩形面积和 1x PBS 体积, 完全覆盖导线的功能部分。使用双面胶带安装在幻灯片上的电线的非功能部分。</li>
	<li>目视检查导线两侧以枚举捕获的单元格 (图 2B 和 2F)。 注意: 细胞的存在是用荧光显微镜来确定的, DAPI 和 FITC 都装有过滤器。导线的两边可以被检查, 并且细胞的数量被评估 (为高细胞数) 或计数 (仅可行, 如果少量细胞被连接到导线)。如果不需要单元格枚举, 则可以跳过此步骤。</li>
	<li>扫描后, 将导线放回包含 1x PBS 的15毫升管 (见2.3.6 的注意事项和第3节), 在黑暗中储存电线。 注意: 大多数的非功能部分的导线可以被切割 (包括弯曲) 和删除放松存储。</li>
</ol>4. 目标细胞的脱离和恢复
注: 分离后应在4小时内完成细胞脱离。
<ol><li>4毫克的释放缓冲组件 (材料和试剂表) 每毫升 1x PBS 和过滤解决方案通过一个0.2 µm 无菌过滤器, 以获得现成使用的缓冲区。 注: 该线的聚合物由水凝胶组成, 其功能是抗 EpCAM 抗体, 允许绑定到 EpCAM 呈现的靶细胞。释放缓冲器含有一种能裂解水凝胶的碳氧水解酶。蛋白水解裂解导致聚合物的降解, 从而使捕获的细胞脱离。</li>
	<li>预热释放缓冲器在37°c 为5分钟。</li>
	<li>添加1.6 毫升释放缓冲, 以填充1.5 毫升的反应管。 注: 为1.5 毫升反应量设计的管允许应用1.6 毫升, 这是必要的淹没的功能部分的电线。</li>
	<li>在37°c (水浴) 的释放缓冲器中孵化出导线的功能部分, 20 分钟. 使用带导线的橡胶帽代替管帽固定1.5 毫升管中的导线, 以避免在水浴中孵化时受到污染。</li>
	<li>将电线/管总成转到 RT 上的振动筛, 500 转15分钟。 注: 振动筛有1.5 毫米的轨道。</li>
	<li>将电线/管组件放置在离心机中, 并将其旋转 300 x g以10分钟。</li>
	<li>从管中取下导线, 关闭瓶盖, 再在 300 x g上离心管10分钟。 注: 电线可以存储在 1x PBS 2-6 °c 在黑暗中的细胞计数, 以评估支队效率/检查的细胞留在电线上。</li>
	<li>为了减少细胞悬浮液的体积, 去除除100µL (微操作系统) 或另一种300µL (cytocentrifugation 和随后的激光显微切割) 的上清, 并立即进行单细胞取样。</li>
</ol>5. 单细胞取样
<ol><li>微操作系统 注: 单个细胞将被添加到2µL 细胞裂解主混合允许 WGA。根据要处理的单元数准备主混合, 计算额外的体积, 以减少由于吹打和地方细胞裂解主混合在冰上。<ol>
<li>根据 Czyż et al18所概述的协议, 准备细胞裂解主混合 (WGA 试剂盒的组件、材料和试剂表)。简单地, 混合2.0 µL 的反应缓冲器, 1.3 µL octylphenoxypolyethoxyethanol (10% 溶液), 1.3 µL 4-(11, 33-tetramethylbutyl) 苯基聚乙二醇 (10% 溶液), 2.6 µL 的蛋白酶 K 溶液, 12.8 µL RNase/DNase 无水获得主组合为10个样品 (总容量20µL)。 注: 有关更多示例, 请相应地增加卷。细胞裂解主成分的组成不同于使用激光显微切割样品的替代程序 (见 5.2)。</li>
		<li>使用油脂笔绘制一个区域, 保持细胞悬浮到位。如果单元格密度太高 (i. e), 请调整区域和音量. 在玻片上标记一个较大的区域, 载入 1x PBS 和细胞悬浮的整除)。</li>
		<li>将整个染色细胞悬浮液吸管 (在步骤4.8 中获得) 放到玻璃滑梯上。</li>
		<li>将玻璃滑梯转移到装有机器人的显微镜上。让单元格结算5分钟 (图 2D 和 2H)。</li>
		<li>准备0.2 毫升的 PCR 管加载2.0 µL 细胞裂解主混合 (见步骤 5.1.1), 并储存在冰上。</li>
		<li>使用机器人在1µL 的 1x PBS 中收集单细胞, 并将细胞转移到含有细胞裂解主混合物的管中。 注: 用于将 micromanipulated 细胞转移到细胞裂解缓冲液中, 将毛细管直接放入溶解溶液的2µL 中, 然后弹出, 直至气泡可见。</li>
		<li>在 2000 x g上简单地向下旋转3秒的样本, 使用桌面离心收集管道底部的所有液体。把样品放在冰上。</li>
		<li>直接进行单单元格 WGA (参见6节和 Czyż et 。19)。</li>
	</ol>
</li>
	<li>激光显微切割 (替代单细胞取样方法: 5.1。微操作系统) 注意: 本节中使用的主混合组合与微操作系统5.1 节中使用的结构不同。单个细胞将被添加到4.5 µL 细胞裂解主混合 (WGA 试剂盒,材料和试剂表的组成部分) WGA。<ol>
<li>根据 Czyż的et 等, 准备细胞裂解主组合。19通过混合5.0 µL 的反应缓冲器, 1.3 µL octylphenoxypolyethoxyethanol (10% 溶液), 1.3 µL 4-(11, 33-tetramethylbutyl) 苯基聚乙二醇 (10% 溶液), µL 的蛋白酶 K 溶液, 2.6 µL RNase/DNase 水获得10样品 (总容积45µL) 的主混合。把主混在冰上。 注: 有关更多示例, 请相应地增加卷。</li>
		<li>紫外线处理膜涂层的幻灯片, 适合激光显微切割。因此, 将幻灯片放置在 DNA 交叉链接器设备中, 并将其暴露在最高紫外线下大约10分钟。</li>
		<li>将膜涂层的幻灯片组装在 cytocentrifuge 中, 并将整个细胞悬浮在 300 x g上 cytospin 5 分钟。 注: 当使用液中系统时, 将单元格 cytocentrifuged 到解决方案中的幻灯片上时, 请在 cytocentrifugation 后取出上清, 并在 1140 x g上应用干燥旋转1分钟。</li>
		<li>直接进行激光显微切割或让 cytospins 空气干燥过夜, 并冻结-20 摄氏度的样品, 长期贮存。</li>
		<li>吸管4.5 µL 细胞裂解大师混合到0.2 毫升 PCR 管的上限和收获单细胞通过激光显微切割。</li>
		<li>从激光显微切割显微镜中提取 PCR 管, 并关闭管。通过短旋转收集管底部的所有液体, 并将样品放在冰上。</li>
		<li>继续进行单细胞 WGA 或将隔离样品储存在-80 摄氏度, 在进一步加工前1月。</li>
	</ol>
</li>
</ol>6. 基于适配器链接器的整个基因组放大18,19
注意: 确保所有必要的主混合 (WGA 套件的组件、材料和试剂表) 都准备就绪并存储在准备使用的冰上。主混合包括 dna 消化混合1为 micromanipulated (见 5.1) 样品 (含2.0 µL 反应缓冲器, 2.0 µL 的MseI 限制酶和16.0 µL RNase/DNase 无水) 或 DNA 消化混合2为激光显微切割 (见 5.2.)样品 (包含2.5 µL MseI 限制酵素和2.5 µL RNase/DNase 水), 预退火大师组合 (包含5.0 µL 反应缓冲器, 5.0 µL 每个 preannealing PCR 适配器和15.0 µL RNase/DNase 无水), 结扎主混合 (包含30.0 µL 预退火 PCR 适配器, 10.0 µL 腺苷磷酸溶液, 10.0 µL T4 连接酶溶液) 和初级 pcr 主混合 (含30.0 µL 的 pcr 缓冲, 20.0 µL 10 毫米核苷酸混合溶液, 10.0 µL 的聚合酶混合和340.0 µL 的 RNase/DNase 无水)。所有卷都提供准备10样品。相应地调整到样本数。
<ol><li>对于细胞裂解, 将 micromanipulated/激光显微切割样品放在热循环仪中, 并通过孵化细胞在42°c 为45分钟, 65 °c 30 分钟和80°c 进行单细胞裂解, 10 分钟。 注: 使用加热盖子的热循环仪。细胞裂解可以做10到15小时的 O/N。在这种情况下, 省略孵化步骤在65摄氏度。</li>
	<li>运行预退火的 PCR 适配器。因此, 在65摄氏度的热循环仪中孵化预退火主混合1分钟后再进一步49循环, 每1分钟, 随着温度逐渐降低1摄氏度/周期。在50个周期 (在15°c) 以后, 孵化大师混合在15°c 直到进一步用途。 注意: 这一步是生成适合于结扎的非对称适配器 (参见步骤 6.6) 的必要步骤, 其中单细胞 DNA 受MseI 限制摘要的约束。</li>
	<li>细胞裂解后, 将裂解样品在 2000 x g上旋转3秒以收集底部的所有液体, 并将其放在冰上。</li>
	<li>为了片段 dna, 添加2µL 的 dna 消化混合1到每个裂解 micromanipulated 样本或0.5 µL 的 dna 消化混合2到激光显微切割样品和运行限制文摘。使用热循环仪的加热盖子在37°c 为5分钟, 其次是限制酶失活步骤65°c 5 分钟。 注: 消化37摄氏度可延长至3h。这是唯一的协议步骤不同之间的样品从微操作系统和样品获得的激光显微切割。</li>
	<li>向下旋转样品, 收集底部的所有液体, 放在冰上。</li>
	<li>为结扎限制被消化的脱氧核糖核酸和预退火的适配器, 增加5.0 µL 结扎大师混合对每个被消化的脱氧核糖核酸样品并且结扎它在热量循环仪在15°c 为 1 h。 注: 此步骤可扩展到 12 h 或执行 O/N。</li>
	<li>向下旋转样品, 收集底部的所有液体, 放在冰上。</li>
	<li>为 WGA 添加40.0 µL 的主要 PCR 主混合到每个结扎产品和运行 WGA 在热循环仪与加热盖子如下: 首先, 孵化的样品在68摄氏度3分钟。第二, 周期为15次, 在94°c 为四十年代, 57 °c 为三十年代和68°c 为1分钟三十年代 +1 s/周期。然后增加9个周期在94°c 为四十年代, 57 °c + 1 °c/周期为三十年代, 68 °c 为1分钟四十五年代 +1 s/周期。此后, 循环23倍于94°c 为四十年代, 65 °c 为三十年代, 68 °c 为1分钟五十三年代 +1 s/周期。最后, 在68摄氏度的3分钟内完成最后的延伸, 并将样品冷却到4摄氏度。</li>
	<li>向下旋转样品, 收集底部的所有液体, 检查 DNA 质量, 或者将样品贮存在-20 摄氏度或-80 摄氏度, 用于长期贮存。</li>
</ol>7. WGA 产品的质量控制
注: 在运行 4plex QC PCR 5后, 根据 DNA 涂片的范围和放大产品的数量检查 WGA 产品的质量。运行 WGA 整除数和各自的 QC-PCR 样品在1.0% 琼脂糖凝胶为评估。
<ol><li>准备用于4plex 质量控制 PCR (表 1) 的所有底漆的100µM 库存解决方案。添加8µL 的每一个底漆到136.0 µL 的 PCR 级水产生200µL 的底漆池。涡流解决方案为 3 s, 旋转它在 2000 x g为 3 s 和整除20µL 为存贮在-20 °c。</li>
	<li>使用标准 pcr 试剂盒, 准备一个多重 pcr 组合, 包括6.0 µL 的 2x pcr 成分 (底漆除外), 4.5 µL 的 pcr 级水和0.5 µL 的底漆池。</li>
	<li>添加1.0 µL 的 WGA 产品, 旋转下来, 并运行 PCR 在94摄氏度2分钟后35循环变性在94摄氏度为1分钟, 底漆退火在56°c 为1分钟和底漆延伸在72°c 为1分钟三十年代和最后的引伸步在72°c 为7分钟冷却它到4°c, 转动它并且放置样品在冰上为凝胶分析同一天或在-20 °c 为以后分析。 注: 冷在热循环仪可以执行在12°c, 以增加寿命的珀尔的元素。</li>
	<li>分析 WGA 产品 (从6.8 获得) 和各自的 4plex QC PCR 产品在琼脂糖凝胶5。</li>
</ol>

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作者没有什么可透露的。

所描述的协议旨在从功能化的导线 (称为设备) 中检索细胞, 用于前体单细胞基因组分析。我们测试了三 EpCAM 阳性细胞系 (HT-29、LNCaP 和 VCaP), 但原则上, 这种方法可应用于任何表达 EpCAM 的细胞系。EpCAM 阳性靶细胞可以呈现为纯细胞悬浮 (见 2.1.) 或混合成多余的背景细胞 (e. g. 外周血, 见 2.2)。特别是后者可能被用来测试在罕见的细胞条件下的隔离能力, 对连接到导线的细胞数量的微调可以使用描述的低容量方法 (见 2.3)。由于细胞系之间的差异是预期的, 适当的细胞密度充电的导线, 旋转速度和孵化时间需要通过评估所附的细胞数量使用免疫荧光显微镜来进行经验测试。
对于基因组下游应用在单细胞水平 WGA, 是必需的。WGA 的选择方法可能不同, 在原则上, 我们的方法是开放的所有方法比可以处理从微操作系统或激光显微切割获得的样品。在本协议中, 我们使用一种基于酶碎片 DNA 的方法, 其性能优于基于 WGA7的热碎片。可选的单个单元格可以被转发到等温 WGA 允许后续的 DNA 分析20,21。
该协议的目的是恢复完整的细胞后, 被附加到新一代功能性导线的基因组单细胞分析。第一代功能性电线, 这也代表了目前在市场上唯一的 CE 批准的体内浓缩装置, 用于枚举转移癌患者的 CTCs。以前的出版物和我们自己的数据表明, 与当前的黄金标准技术2相比,体内隔离技术更敏感。因此, 将此体内隔离技术与设备中实现的恢复功能配合使用, 可以将高反恐恢复与单单元级的特性结合起来。
然而, 该设备没有清除, 以用于病人, 因此, 临床数据是不可用的。前体应用程序 (即在取样后从外周血中隔离 CTCs) 不推荐, 因为该技术适应了在肘中存在的设置,如低直径的徒劳 (增加概率直接接触 CTCs 和捕捉抗体) 和长期保留时间 (30 分钟, 允许2-3 升血液通过导线)。但是, 如第2.2 节所述。还可以从混合示例7中隔离目标单元格。
目前该方法的局限性是从导线脱离后, 无固定细胞恢复效率低下。然而, 在分离治疗之前, 细胞固定步骤可能会改善这一点。
总之, 此协议是迈向将体内隔离技术与细胞恢复用于基因特征的单个细胞的联合使用的第一步。进一步的努力需要解决细胞恢复和清除在患者中的应用的优化1,2。然而, 治疗监测和治疗决策的个性化药物 CTCs。因此, 这种方法是迈向基因组反恐小组在单细胞水平上的第一步。
当完整的细胞被收获时, 对单细胞转录分析的应用似乎是可行的。但是, 仍有待评估恢复过程是否对 RNA 完整性产生影响 (例如转发恢复的单个单元格以直接裂解, 其次是 RT qPCR22)。如果成功, 则可以将单个单元格 (例如CTCs) 的特征转移到转录级别, 从而允许进行更详细的分析。在这方面, rna 序列使用 Smart-seq2 提供了一个宝贵的工具, rna 下游分析单细胞作为放大 cDNA (获得在 RNA 序列库准备) 可以受到定量 PCR。这将允许对单个恢复的单元格进行联合目标和基于筛选的分析22,23。
目前的功能性电线是基于抗 EpCAM 抗体, 这是一个广泛使用的上皮标记为阳性选择的 CTCs24。由于一些 CTCs 可能 downregulate 上皮标记, 如细胞角蛋白或EpCAM25, 添加诸如 HER2/new 这样的抗体将增加反恐委员会隔离的几率。通过对几种基于抗体的富集策略进行了研究和评述. 在 2016年26中。固定在导线上的抗体的混合物可能是未来技术的改进, 导致分离更高的数量和其他亚型的 CTCs。
这是强制性的所有步骤涉及导线处理, 以保持线的功能部分淹没在解决方案, 以保持其功能。我们建议在评估时将导线放置在 1x PBS 中 (显微镜;e. g. 步骤3.1。-3.3)和存储。为了避免不适当的染色, 我们建议使用新的染色液在步骤1.2.1。和1.2.2。弱染色的细胞阻碍细胞计数的导线, 可能导致低估的附加细胞。
当插入巴斯德吸管中的丝时, 必须避免堵塞巴斯德吸管与橡胶帽, 以便随后加载细胞悬浮液 (步骤 2.3.4)。橡胶帽用于保持导线到位, 使功能部分位于巴斯德吸管的尖端内。如果盖帽太牢固地附有对巴斯德吸管的后方, 细胞悬浮的装载不是可能的。在这种情况下, 减轻瓶盖, 使被加载的细胞悬浮所取代的空气可以离开吸管。
弯曲导线是关键的它的方向在细胞计数在步骤3.2。和3.3。使用胶带安装电线, 使导线的非功能端向一侧说谎。扫描后的整个功能部分的长度, 电线是轧制 180°, 以便另一半的功能线可以扫描。这一步骤对于初步实验评估导线上的细胞数量是很重要的。一旦计算出特定单元格行和过程的单元格数, 就可以重复该过程而不进行单元格计数 (e. g). 仅检查是否存在单元格或省略此步骤)。仔细的吹打是至关重要的, 以避免细胞丢失时, 减少在步骤4.8 的上清的体积。确保吹打顺利完成, 不造成颗粒的动荡。

最近, CellCollector DC01, 一个医疗线功能性与抗 EpCAM 抗体, 以隔离 CTCs 从外周血的癌症患者, 增加到系统化频谱的反恐委员会枚举1,2,3.在目前正在进行的非转移性前列腺癌研究中, 这一功能性导线报告的患者几乎两倍于反恐委员会阳性患者的阳性和更高的反恐组计数, 与 CellSearch 相比, 反恐委员会枚举中的金标准为3.由于这种令人鼓舞的表现, 从功能性医疗导线中分离细胞进行单细胞分析是可取的, 但无论是用细胞脱离解决方案 (例如胰蛋白酶) 的酶治疗, 还是激光显微切割都不允许恢复完整的单元格 (未显示数据)。
为了让捕获的细胞脱离, 新一代的功能性导线配备了特定的聚合物。这种聚合物, 将捕获抗体链接到导线, 容易受到释放缓冲治疗, 从而使完整细胞 (CellCollector DC03 称为设备) 脱离。新的功能性设备, 允许分离的目标细胞从不同浓度的癌细胞线细胞被注入牛血清白蛋白 (BSA)/磷酸盐缓冲生理盐水 (PBS) 和外周血, 分别。
为了减轻对设备和恢复后细胞的视觉检测, 目标癌细胞被标记为羧基琥珀酰亚胺酯 (CFSE) 和在恢复治疗前的 DNA 染色 (i. e. 充电和分离)。WGA 通过质量控制 PCR4,5, 阵列全息图6,7和目标排序7显示没有区别, 以前评估了治疗对单细胞 DNA 质量的影响与未处理的细胞 micromanipulated 的细胞悬浮物相比,7。这种方法的优点在于将稀有的靶细胞预富集与细胞的恢复结合在一起进行单细胞下游分析 (图 1)。当前 CE 标记的体内集合设备通常用于枚举 CTCs, 而不是用于单细胞分子特性2,8。然而, 更全面的分析, 以调查 CTCs 之间的异质性长的分析在单个细胞水平 (即目标排序在单细胞水平)。其他基于细胞的方法是基于磁隔离的 EpCAM 阳性 CTCs 和单细胞处理基于介电泳的后续分子遗传学分析 9, 10.CTCs 的分子特征是其在临床环境中有效实施的重要要求, 在转移性叶栅的基础研究中同样重要。与 CTCs 平行, 循环肿瘤 dna (ctDNA) 已成为非常重要的, 因为它允许 DNA 分析的肿瘤负担与最小的技术隔离程序11,12。基于单元格的方法可以作为一个互补的贡献, 因为它允许 RNA13,14和蛋白质15表达式分析, 也为反恐委员会派生细胞培养或移植16,17. 虽然仍然需要克服诸如低细胞恢复和清除对患者使用的障碍, 但捕捉和释放方法对稀有靶细胞的定性具有重要的下一步。

<table><tbody><tr><td>Gilupi Release Buffer</td><td>Gilupi</td><td>GIL083</td><td>Used to detach cells from the wire</td></tr><tr><td>McCoy&#039;s 5A Medium (1x) suppl. with L-Glutamine</td><td>Thermo Fisher Scientific</td><td>16600-082</td><td>Culture medium used for HT-29 cells, adapt culture medium to cell line used for the experiments</td></tr><tr><td>HEPES Buffer Solution (1 M)</td><td>PAA</td><td>S11-001</td><td>Supplement for McCoy&#039;s 5A Medium</td></tr><tr><td>Foetal Bovine Serum Gold</td><td>PAA</td><td>A15-151</td><td>Supplement for McCoy&#039;s 5a Modified Medium</td></tr><tr><td>Penicillin-Streptomycin (100x)</td><td>Biowest</td><td>L0018-100</td><td>Supplement for McCoy&#039;s 5a Modified Medium</td></tr><tr><td>Phosphate Buffered Saline (1x PBS)</td><td>Thermo Fisher Scientific</td><td>10010-015</td><td></td></tr><tr><td>Accutase</td><td>Biowest</td><td>L0950-100</td><td>Cell detachment solution (cell culture)</td></tr><tr><td>Water bath</td><td>GFL</td><td>Type 1003</td><td>Used for prewarming solutions</td></tr><tr><td>Incubator</td><td>Thermo Fisher Scientific</td><td></td><td>Used to incubate cells (cell culture)</td></tr><tr><td>50 mL reaction tubes</td><td>VWR</td><td>525-0403</td><td></td></tr><tr><td>Roller mixer</td><td>Stuart Scientific</td><td>SRT6D</td><td>Used to attach cells to the wire while keeping the cells from sedimenting</td></tr><tr><td>CellTrace CFSE Cell Proliferation Kit</td><td>Thermo Fisher Scientific</td><td>C34554</td><td>Used to label living cells (cytoplasmic label)</td></tr><tr><td>Hoechst 33342</td><td>H3570</td><td>Thermo Fisher Scientific</td><td>Used to stain DNA (nucleic counterstain)</td></tr><tr><td>Centrifuge (equipped for holding 15 mL and 50 mL reaction tubes)</td><td>Thermo Fisher Scientific</td><td>Heraeus Megafuge 40R</td><td>Used for pelleting cells</td></tr><tr><td>Observer Z1 (Fluorescence/laser microdissection microscope)</td><td>Carl Zeiss Microimaging</td><td></td><td>Inverted (immunofluorescence) microscope capable of laser microdissection; Fluorescence microscopes must be able to detect Hoechst 33342 (DAPI filter) and CFSE (FITC filter)</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A4503-50G</td><td>Used for coating glass/plastic surfaces</td></tr><tr><td>MS1 Minishaker</td><td>Sigma-Aldrich</td><td>Z404039</td><td>Used for vortexing</td></tr><tr><td>Vacutainer EDTA (or Na-heparin) tubes</td><td>BD</td><td>366450 (or 366480)</td><td>Used as reaction tube for ex vivo capture of target cells</td></tr><tr><td>Detektor CANCER03 DC03</td><td>Gilupi</td><td>GIL003</td><td>Functionalized wire capable of isolating and detaching EpCAM-positive cells (also referred to as catch&amp;amp;release, C&amp;amp;R)</td></tr><tr><td>Syringe needles (G20)</td><td>VWR</td><td>613-0554</td><td>Used to puncture rubber caps in order to allow the non-functional part of the C&amp;amp;R to lead through it</td></tr><tr><td>Neubauer chamber</td><td>Roth</td><td>T729.1</td><td>Used to count cells</td></tr><tr><td>Pasteur pipettes 150 mm</td><td>Volac</td><td>D810</td><td>Used for the low-volume application of cells to the C&amp;amp;R</td></tr><tr><td>Grease pen</td><td>Dako</td><td>S2002</td><td>Used on glass slides to hold cell suspension in place</td></tr><tr><td>Steril syringe filter (0.2 &amp;micro;m)</td><td>Corning</td><td>431219</td><td>For filtering freshly prepared Gilupi Release Buffer</td></tr><tr><td>Delfia PlateShaker</td><td>Perkin Elmer</td><td>1296-003</td><td>Used for agitation during cell detachment</td></tr><tr><td>1.5 mL tubes</td><td>Eppendorf</td><td>30,125,150</td><td></td></tr><tr><td>Ampli1 WGA Kit</td><td>Silicon Biosystems</td><td></td><td>To be ordered via Silicon Biosystems</td></tr><tr><td>Axiovert M200 equipped with Mikromanipulator MMJ and CellTram vario</td><td>Zeiss/Eppendorf</td><td></td><td>Use micromanipulation at hand</td></tr><tr><td>Microcapillaries (25 &amp;micro;m in diameter), CustomTip Type I</td><td>Eppendorf</td><td>930001201</td><td>Used for micromanipulating cells</td></tr><tr><td>0.2 mL PCR tubes</td><td>Biozym</td><td>710920</td><td></td></tr><tr><td>Cytocentrifuge and equippment</td><td>Hettich</td><td>Universal 32</td><td>Use cytocentrifuge at hand</td></tr><tr><td>Thermo cycler</td><td>Bio-Rad</td><td>DNA Engine Dyad</td><td>Every thermo cycler with heated lid can be used</td></tr><tr><td>Microcentrifuge</td><td>Roth</td><td>CX73.1</td><td>Use desk-top centrifuge at hand</td></tr><tr><td>MembraneSlide 1.0 PET</td><td>Zeiss</td><td>415101-4401-050</td><td>Membrane-coated glass slides used for laser microdissection</td></tr><tr><td>UV Stratalinker 1800</td><td>Stratagene</td><td>400072</td><td>Use DNA cross linker at hand</td></tr><tr><td>Standard PCR reagents</td><td></td><td></td><td></td></tr><tr><td>6x DNA Loading</td><td>Thermo Fisher Scientific</td><td>R0611</td><td>Use gel loading dye at hand</td></tr><tr><td>DNA ladder</td><td>BioLabs</td><td>N0551G</td><td>Use 100 bp ladder at hand</td></tr><tr><td>Agarose</td><td>Biozym</td><td>840004</td><td></td></tr><tr><td>Gel electorphoresis station</td><td>Bio-Rad</td><td></td><td>Use electrophoresis system at hand</td></tr><tr><td>Gel Red Nucleic Acid Stain</td><td>Biotium</td><td>41003</td><td>Intercalating dye for DNA visualization In agarose gels</td></tr></tbody></table>

target cell pre-enrichment and whole genome amplification for single cell downstream characterization

在非靶细胞的高背景下, 利用靶细胞表面蛋白特异的抗体, 可以分离出稀有靶细胞。最近开发的方法使用医疗导线功能性与抗上皮细胞黏附分子 (EpCAM) 抗体为在体内隔离循环肿瘤细胞 (CTCs)1。在非转移性前列腺癌患者匹配队列表明,体内隔离技术导致患者阳性率高于 CTCs, 以及反恐委员会列举的现行金标准相比较高。由于细胞无法从目前的医疗设备中恢复, 一种新的功能性导线 (称为装置) 是制造的, 允许捕获和随后的细胞分离通过酶处理。细胞被允许连接到该设备, 可视化显微镜和分离使用酶治疗。回收的细胞被 cytocentrifuged 到膜涂层的幻灯片上, 并通过激光显微切割或微操作系统来单独收割。单细胞样本然后接受单细胞全基因组放大, 允许多种下游分析, 包括筛选和目标特定的方法。分离和恢复的过程从单细胞中产生高质量的 DNA, 并不会损害后续的整个基因组放大 (WGA)。单个细胞的扩增 DNA 可以被转发到筛查和/或靶向分析, 如阵列比较基因组杂交 (阵列全息图) 或测序。该设备允许从人工稀有细胞样本 (即500 靶细胞被注入5毫升外周血) 中分离出前体。虽然细胞的脱离率是可以接受的 (50-90%), 分离的细胞的恢复率在幻灯片跨广泛的范围依赖于细胞线使用 (< 10-> 50%) 和需要进一步注意。此设备未清除以供患者使用。

CFSE 和核酸染色后的细胞可以用免疫荧光显微镜 (DAPI 和 FITC 过滤器) 进行评估。细胞核呈明亮的 counterstain 在 DAPI 通道, 而细胞细胞质显示统一标签与 CFSE 的异构细胞间强度 (图 2A和2E)。从连接到导线的单元格 (图 2B和2F) 以及从线上分离并在幻灯片上恢复 (图 2D和2H), 预期形状和染色强度类似。
高细胞密度 (例如> 1 000 000 细胞/毫升) 可能导致在充电时导线与细胞的总覆盖。然而, 较低的细胞密度, 在孵化过程中的旋转速度的变化, 和使用不同的细胞系影响的隔离效率和需要单独测试。当5毫升的 HT-29 细胞在 100 000 细胞/毫升上孵化时, 如2.1 节所述, 在导线上捕捉到了254个 * 103 个细胞 (n = 5) 的平均值。用 LNCaP 细胞进行相同的实验, 得到每根导线的平均 440 319 细胞 (n = 5)。
呈现 500, 5 000, 50 000 HT-29 细胞的背景下5毫升的外周血到电线 (根据协议第 2.2.) 导致捕获 75 @ 18 细胞, 25 @ 9 细胞, 和 47 @ 57 细胞 (n = 3, 每个), 分别。如果导线被充电以15µL 细胞悬浮 (1 000 000 细胞/mL) 根据协议在 2.3., 最多 1 000 (HT-29) 细胞可能连接到导线。
细胞分离效率从81% 在 HT-29 细胞 (范围 54.9%-93.2% 和 n = 5) 到 81% (范围 63.51%-92.87%, n = 6) 和 91% (范围 84.7%-95.3%, n = 6) 在 LNCaP 和 VCaP 细胞, 分别。同样, 分离细胞的恢复也不同于细胞系: 由 cytocentrifugation 恢复28% 分离 HT-29 细胞的平均值。而 LNCaP 回收率低于 10%, VCaP 细胞平均回收率为55%。
约75% 的单细胞受 WGA 产量高品质的 WGA 产品: 这是 1) 涂片 WGA 产品范围从0.1 到 > 1 kb (图 4, 顶板) 和 2) 三或四波段在4plex 质量控制 PCR (图 4, 底部面板).WGA 产品可用于 1) 阵列全息分析, 满足单单元阵列-全息图配置文件6,7和 2) 的质量标准,在重新放大 WGA 7后将其定向到。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56394/56394fig1.jpg"> 图 1: 用于隔离和恢复单单元分析的目标单元格的工作流.(A)单元格被培养为90% 融合和(B) , 以获得单细胞悬浮。在用 CFSE 和核酸染色后, (C)功能性导线在5毫升的细胞中使用反应管或在低体积内使用巴斯德吸管尖端孵化。后者允许应用细胞悬浮容量低到15µL. (D)单元格需要在充电后4小时内分离。因此, 该导线被放置在一个1.5 毫升的反应管填充释放缓冲。在摇杆和10分钟离心15分钟的孵化后, 导线被移除, 细胞悬浮离心细胞。(E)恢复的单元格被转移到单细胞微操作系统 (顶部) 或 cytocentrifuged 的玻璃滑块上, 并转到膜涂层的幻灯片上, 并转发到激光显微切割 (底部)。(F)最后, 通过全基因组扩增 (WGA), 将收获的单细胞放大。WGA 产品与阵列全息图和目标下游分析相兼容。EpCAM 表位: 细胞表面的绿色圆圈。功能线: 设备, 灰色三螺旋线。高分子: 紫色的线条。抗 EpCAM 抗体: 橙色。释放缓冲区活动: 红色箭头。油脂标记保持细胞悬浮在地方: 深蓝色椭圆。恢复的细胞悬浮: 浅蓝色。微操作系统毛细管: 黑线。放大: 恢复细胞由微操作系统, cytospin 在膜覆盖的幻灯片包含恢复的细胞 (灰色填充椭圆) 收获。放大: 恢复的细胞是激光 microdissected (灰色圆线: 膜从膜幻灯片作为主干为激光显微切割), 目标与激光束 (蓝线接近膜幻灯片从下面)。<a href="https://cloudflare.jove.com/files/ftp_upload/56394/56394fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56394/56394fig2.jpg"> 图 2: 可视化标记的单元格.(A, E)细胞在充电前: 细胞被标记与 CFSE 和复染与脱氧核糖核酸锂染料显示一系列 CFSE (绿色) 信号强度。(B,F)被标记的细胞被捕获在电线上。(C,G)线在细胞分离过程以后。(D,H)细胞从电线中分离, 通过 cytocentrifugation 恢复到幻灯片上。CFSE: FITC 通道 (绿色)。DNA 染色: DAPI 通道 (蓝色)。<a href="https://cloudflare.jove.com/files/ftp_upload/56394/56394fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56394/56394fig3.jpg"> 图 3: 电线和存储舱.电线的(A)隔间。(B)功能性部件的详细信息。<a href="https://cloudflare.jove.com/files/ftp_upload/56394/56394fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56394/56394fig4.jpg"> 图 4: 从单个 micromanipulated 单元获得的 WGA 产品的质量控制.顶部面板: 从单个细胞获得的 WGA 产品通常会导致 DNA 涂片范围从 0.2 kb 到 > 1.0 kb, 峰值强度在 0.5 kb 左右。样本数 1, 7 和 13 (用星号表示) 显示不优或没有 DNA 涂片。底部面板: WGA 产品有足够的质量, 如果 4plex pcr 产生三或四四 pcr 产品 (在 100, 200, 300, 和 400 bp)。样本1、7、10和13显示少于三个波段, 将被排除在进一步分析之外。车道 M 包含 100 bp 梯子和星号表明 WGA 产品质量不足的样品。<a href="https://cloudflare.jove.com/files/ftp_upload/56394/56394fig4large.jpg" target="_blank">请单击此处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>底漆序列</td>
			<td>注意</td>
		</tr>
<tr>
<td>5′-gtt 国家评估 cc-3</td>
			<td>100 bp 向前底漆</td>
		</tr>
<tr>
<td>5′ gaa ggt gg-3</td>
			<td>100 bp 反向底漆</td>
		</tr>
<tr>
<td>5′-agg tgg agc gct agc-3</td>
			<td>200 bp 向前底漆</td>
		</tr>
<tr>
<td>5′-ttt tgc ggt gga</td>
			<td>200 bp 反向底漆</td>
		</tr>
<tr>
<td>5′ agg gct gg-3</td>
			<td>300 bp 向前底漆</td>
		</tr>
<tr>
<td>5′ gcg tc-3</td>
			<td>300 bp 反向底漆</td>
		</tr>
<tr>
<td>5′《猫海湾合作协定》 gc-3</td>
			<td>400 bp 向前底漆</td>
		</tr>
<tr>
<td>5′-gct agt ggt 增值税 tg-3</td>
			<td>400 bp 反向底漆</td>
		</tr>
</tbody></table>表 1: 4plex 质量控制 PCR 引物序列

Watch this Scientific Journal Video about Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization at JoVE.com

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

该协议是从非目标背景细胞的混合物中恢复和制备稀有靶细胞, 用于单细胞水平的分子遗传表征。DNA 质量等于未处理的单个细胞, 并允许单细胞应用 (筛查和靶向分析)。

单细胞下游特征的靶细胞预富集与全基因组扩增

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

target-cell-pre-enrichment-whole-genome-amplification-for-single-cell

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

8.8K 次观看.  Medical University of Graz. 该协议是从非目标背景细胞的混合物中恢复和制备稀有靶细胞, 用于单细胞水平的分子遗传表征。DNA 质量等于未处理的单个细胞, 并允许单细胞应用 (筛查和靶向分析)。

视频: Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

单细胞测序拷贝数改变的检测

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

科研

遗传学

Transcriptome Analysis of Single Cells

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in culture.1,2,3 In the case of the brain, regions such as the hippocampus contain a complex arrangement of different cell types, each with distinct mRNA profiles. The ability to harvest single cells allows for a more in depth investigation into the molecular differences between and within cell populations. We describe a simple and rapid method for harvesting cells for further processing. Pipettes often used in electrophysiology are utilized to isolate (using aspiration) a cell of interest and conveniently deposit it into an Eppendorf tube for further processing with any number of molecular biology techniques. Our protocol can be modified for the harvest of dendrites from cell culture or even individual cells from acute slices.
We also describe the aRNA amplification method as a major downstream application of single cell isolations. This method was developed previously by our lab as an alternative to other gene expression analysis techniques such as reverse-transcription or real-time polymerase chain reaction (PCR).4,5,6,7,8 This technique provides for linear amplification of the polyadenylated RNA beginning with only femtograms of material and resulting in microgram amounts of antisense RNA. The linearly amplified material provides a more accurate estimation than PCR exponential amplification of the relative abundance of components of the transcriptome of the isolated cell. The basic procedure consists of two rounds of amplification. Briefly, a T7 RNA polymerase promoter site is incorporated into double stranded cDNA created from the mRNA transcripts. An overnight in vitro transcription (IVT) reaction is then performed in which T7 RNA polymerase produces many antisense transcripts from the double stranded cDNA. The second round repeats this process but with some technical differences since the starting material is antisense RNA. It is standard to repeat the second round, resulting in three rounds of amplification. Often, the third round in vitro transcription reaction is performed using biotinylated nucleoside triphosphates so that the antisense RNA produced can be hybridized and detected on a microarray.7,8

In this article we describe a simple method for the harvesting of single cells from rat primary neuronal cultures and subsequent transcriptome analysis using aRNA amplification. This approach is generalizable to any cell type.

单细胞的转录组学分析

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in ...

神经科学

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is inherently a single-cell assay, however prior to molecular analysis, the target cells are often prospectively isolated in bulk, thereby losing single-cell resolution. Single-cell gene expression analysis provides a means to understand molecular differences between individual cells in heterogeneous cell populations. In bulk cell analysis an overrepresentation of a distinct cell type results in biases and occlusions of signals from rare cells with biological importance. By utilizing FACS index sorting coupled to single-cell gene expression analysis, populations can be investigated without the loss of single-cell resolution while cells with intermediate cell surface marker expression are also captured, enabling evaluation of the relevance of continuous surface marker expression. Here, we describe an approach that combines single-cell reverse transcription quantitative PCR (RT-qPCR) and FACS index sorting to simultaneously characterize the molecular and immunophenotypic heterogeneity within cell populations.
In contrast to single-cell RNA sequencing methods, the use of qPCR with specific target amplification allows for robust measurements of low-abundance transcripts with fewer dropouts, while it is not confounded by issues related to cell-to-cell variations in read depth. Moreover, by directly index-sorting single-cells into lysis buffer this method, allows for cDNA synthesis and specific target pre-amplification to be performed in one step as well as for correlation of subsequently derived molecular signatures with cell surface marker expression. The described approach has been developed to investigate hematopoietic single-cells, but have also been used successfully on other cell types.
In conclusion, the approach described herein allows for sensitive measurement of mRNA expression for a panel of pre-selected genes with the possibility to develop protocols for subsequent prospective isolation of molecularly distinct subpopulations.

Bulk gene expression measurements cloud individual cell differences in heterogeneous cell populations. Here, we describe a protocol for how single-cell gene expression analysis and index sorting by Florescence Activated Cell Sorting (FACS) can be combined to delineate heterogeneity and immunophenotypically characterize molecularly distinct cell populations.

一种组合单细胞法表征人茎和祖种群的分子和免疫表型异质性

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is ...

Purification of Low-abundant Cells in the Drosophila Visual System

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small numbers of cells. Utilizing this technology to study development in the Drosophila visual system, which boasts a wealth of cell type-specific genetic tools, provides a powerful approach for addressing the molecular basis of development with precise cellular resolution. For such an approach to be feasible, it is crucial to have the capacity to reliably and efficiently purify cells present at low abundance within the brain. Here, we present a method that allows efficient purification of single cell clones in genetic mosaic experiments. With this protocol, we consistently achieve a high cellular yield after purification using fluorescence activated cell sorting (FACS) (~25% of all labeled cells), and successfully performed transcriptomics analyses on single cell clones generated through mosaic analysis with a repressible cell marker (MARCM). This protocol is ideal for applying transcriptomic and genomic analyses to specific cell types in the visual system, across different stages of development and in the context of different genetic manipulations.

Here, we present a cell dissociation protocol for efficiently isolating cells present at low abundance within the Drosophila visual system through fluorescence activated cell sorting (FACS).

果蝇视觉系统中低丰细胞的纯化

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small ...

Reusable Single Cell for Iterative Epigenomic Analyses

Current single-cell epigenome analyses are designed for single use. The cell is discarded after a single use, preventing analysis of multiple epigenetic marks in a single cell and requiring data from other cells to distinguish signal from experimental background noise in a single cell. This paper describes a method to reuse the same single cell for iterative epigenomic analyses. 
In this experimental method, cellular proteins are first anchored to a polyacrylamide polymer instead of crosslinking them to protein and DNA, alleviating structural bias. This critical step allows repeated experiments with the same single cell. Next, a random primer with a scaffold sequence for proximity ligation is annealed to the genomic DNA, and the genomic sequence is added to the primer by extension using a DNA polymerase. Subsequently, an antibody against an epigenetic marker and control IgG, each labeled with different DNA probes, are bound to the respective targets in the same single cell. 
Proximity ligation is induced between the random primer and the antibody by adding a connector DNA with complementary sequences to the scaffold sequence of the random primer and the antibody-DNA probe. This approach integrates antibody information and nearby genome sequences in a single DNA product of proximity ligation. By enabling repeated experiments with the same single cell, this method allows an increase in data density from a rare cell and statistical analysis using only IgG and antibody data from the same cell. The reusable single cells prepared by this method can be stored for at least a few months and reused later to broaden epigenetic characterization and increase data density. This method provides flexibility to researchers and their projects.

The present protocol describes a single-cell method for iterative epigenomic analyses using a reusable single cell. The reusable single cell allows analyses of multiple epigenetic marks in the same single cell and statistical validation of the results.

用于迭代表观基因组分析的可重复使用的单细胞

Current single-cell epigenome analyses are designed for single use. The cell is discarded after a single use, preventing analysis of multiple epigenetic ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

检测和罕见富集抗原特异性B细胞的表型和功能分析

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

免疫与感染

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

单细胞基因组测序的超高通量微流控平台

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

生物工程

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 ...

生物学

小鼠颈动脉的两步细胞消化

Preparation of a Single-Cell Suspension from Mouse Carotid Arteries for Single-Cell Sequencing

Carotid arteries are major blood vessels in the neck that supply blood and oxygen to the brain, but carotid stenosis occurs when carotid arteries are clogged by plaque. Revealing the cellular composition of the carotid artery at the single-cell level is essential for treating carotid atherosclerosis. However, there is no ready-to-use protocol for the preparation of single-cell suspensions from carotid arteries. To obtain a suitable protocol for the dissociation of normal carotid arteries at the single-cell level with less damage to cells, we designed a two-step digestion method by integrating the digestion process of collagenase/DNase and trypsin. Acridine orange/propidium iodide (AO/PI) dual-fluorescence counting was used to detect cell viability and concentration, and it was found that the single-cell suspension satisfied the requirements for single-cell sequencing, with the viability of cells over 85% and a high cell concentration. After single-cell data processing, a median of ~2500 transcripts per cell were detected in each carotid artery cell. Notably, a variety of cell types of the normal carotid artery, including vascular smooth muscle cells (VSMCs), fibroblasts, endothelial cells (ECs), and macrophages and dendritic cells (M&#966;/DCs), were concurrently detectable. This protocol may be applied to prepare a single-cell suspension of blood vessels from other tissues with appropriate modifications.

作者聚焦：血管组织解离和探索用于靶向治疗的单细胞亚簇 

Here we describe a two-step cell digestion protocol for preparing a single-cell suspension of mouse carotid arteries.

从小鼠颈动脉制备用于单细胞测序的单细胞悬液

Carotid arteries are major blood vessels in the neck that supply blood and oxygen to the brain, but carotid stenosis occurs when carotid arteries are ...

单细胞下游特征的靶细胞预富集与全基因组扩增

摘要

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此视频中的章节

单细胞测序拷贝数改变的检测

单细胞的转录组学分析

一种组合单细胞法表征人茎和祖种群的分子和免疫表型异质性

果蝇视觉系统中低丰细胞的纯化

用于迭代表观基因组分析的可重复使用的单细胞

检测和罕见富集抗原特异性B细胞的表型和功能分析

单细胞基因组测序的超高通量微流控平台

基于滴瓣的成年哺乳动物组织单细胞转录瘤

从小鼠颈动脉制备用于单细胞测序的单细胞悬液

从小鼠颈动脉制备用于单细胞测序的单细胞悬液