我们感谢 Rhee 实验室的成员分享未发布的数据和宝贵的讨论。这项工作得到了加拿大自然科学和工程研究理事会（NSERC）赠款RGPIN-2018-06404（H.R.）的支持。

注：建议自动提取蒸馏和去离子化水（ddH2O）制作缓冲液和反应大师混合。第1-4节描述细胞裂解和声波化，第5-7节描述色素免疫沉淀（ChIP），第8-11节描述珠子上的酶反应，第12节和第13节描述ChIP精英和DNA纯化，第14-19节描述库准备。
1. 收获和交叉链接细胞
<ol><li>将小鼠 ES 细胞分化为后线粒体神经元后，在收获的细胞中加入 11% 甲醛，最终浓度为 1%（v/v）。在室温下（RT，25°C），摇动平台上的岩石单元（摇动器、摇床或旋转器）为 15 分钟。 注：根据要交叉链接的目标蛋白质，甲醛交叉链接较少（例如，最终浓度为 0.5%）或双交叉链接与脱硫谷氨酸（DSG）可以使用。</li>
	<li>将 2.5 M 甘氨酸添加到 150 mM 的最终浓度中，以阻止交叉链接反应。Rt 的摇动平台上的岩石细胞， 5 分钟。</li>
	<li>离心在RT的15 mL圆锥管的交叉链接细胞，6分钟，在1，350 x g.吸气溶液，然后在5 mL的1x磷酸盐缓冲盐水（PBS）中重新悬生细胞。 注：交叉链接的细胞颗粒在液氮闪光冻结后可储存在-80°C。</li>
</ol>2. 细胞裂解
注：此协议中的以下步骤是大约 2 x 107 神经元细胞与小鼠 ES 细胞分化。为了打破开放的细胞，将使用含有各种洗涤剂的裂解缓冲器。在使用前将 1000 倍全蛋白酶抑制剂 （CPI） 库存添加到 50 mL 缓冲区。
<ol><li>准备裂解缓冲区 1+3，如 表 2中所述。</li>
	<li>在冰上解冻交叉链接的细胞颗粒，然后在3 mL的裂解缓冲区1在15 mL圆锥管中彻底重新悬生细胞颗粒。在4°C下在摇动平台上摇滚15分钟。离心机在 1，350 x g 5 分钟在 4 °C 和吸气超纳坦。</li>
	<li>在3 mL的裂解缓冲区2中彻底重新悬念颗粒细胞。在4°C下在摇动平台上摇滚10分钟。离心机在 1，350 x g 5 分钟在 4 °C 和吸气超纳坦。</li>
	<li>在每个颗粒中加入1 mL的裂解缓冲区3，然后保持在冰上。立即进行声波。</li>
</ol>3. 声波色素
注意：在此声波过程中将样品放在冰上或在 4 °C 下，以减少交叉链接反转。
<ol><li>使用无菌铲，在 15 mL 聚苯乙烯管上添加声波珠（材料表）高达 0.2 mL 毕业标记。在 1x PBS 的 600 μL 中通过涡流洗涤声波珠，直到看不到干燥点。在 30 x g 时将管子离心 5 s，吸气 1x PBS。 注：聚苯乙烯管中的声波比聚丙烯管中的声波效率更高。如果少量的细胞（例如，+lt;106 细胞）被声波化，则使用 1.5 mL 聚苯乙烯管，而无需添加声波珠。</li>
	<li>在 1 mL 裂解缓冲区 3（从步骤 2.4）中彻底重新悬生核裂解，然后转移到含有声波珠的 15 mL 聚苯乙烯管中。短暂地旋转聚苯乙烯管。</li>
	<li>要将交叉链接的染色质DNA分割开来，在4°C下进行核裂解，以获得优化的周期数，功率振幅为30W，声波周期设置为30s on/30关闭。 注：优化每个细胞类型和批次的声波将产生最佳的ChIP-exo产量。对于 2 x 107 小鼠神经元细胞，上述设置的 20+30 声波周期将分裂色素在 100-500 bp 范围内。</li>
	<li>声波完成后，将所有超纳坦（声波化解石）从每个样品转移到1.5 mL管。在每个样品中加入 10% 2-+4-（2，4，4-三乙基苯丙胺-2-yl）苯氧乙醇，最终浓度为 1%。通过管道混合。</li>
	<li>对于颗粒细胞碎片，离心机样品在 13，500 x g 10 分钟，在 4 °C。 将所有声波化的利酸盐（超纳坦剂）从每个样品转移到新的1.5 mL管。</li>
	<li>从每个样品中取出 30 μL 的声波化利酸盐（约 3% 的声波解石），并加入新的 1.5 mL 管以检查声波。将剩余的声波解质在 4 °C 下存放，直到准备好用抗体涂层珠子孵育。</li>
</ol>4. 检查声波
<ol><li>通过添加 2 mL 的 0.5 M 特里斯-HCl （pH 8.0）， 2 mL 的 0.5 M EDTA， 10 mL 的 10% 硫酸钠 （SDS） 和 6 mL 的自动包装 ddH 2 O， 使 20 mL 的2xl 2倍蛋白酶 K 缓冲区。不要将 CPI 添加到此缓冲区中。存储在RT的50 mL管。</li>
	<li>要逆转蛋白质-DNA交叉链接，通过去除蛋白质，从每个样本（从步骤3.6）中取出30μL的声波染色质，添加166μL的自动夹式ddH2O， 200 μL的2倍蛋白酶K缓冲区和4μL的20毫克/mL蛋白酶K.短暂漩涡样品，然后在65°C孵育1~3小时在24 x g。</li>
	<li>注：声波分析可以在65°C下在夜间反向交叉连接。</li>
	<li>使用PCIA（25：24：1）和乙醇沉淀方法提取DNA如下。 警告：PCIA是有毒的。使用带标准个人防护设备 （PPE） 的烟雾罩下。<ol>
<li>在 1.5 mL 管中将 400 μL 的 PCIA 添加到每个样品中，将涡流设置为最大速度，并将涡流样本设置为 20 秒。离心机在 18，400 x g 6 分钟在 Rt 。将观察两个阶段。小心地将每个样品的上一层（透明相）转移到新的1.5 mL管中。</li>
		<li>在 37 °C 下加入 1 μL 的 10 毫克/mL RNase A. 孵化样品 30 分钟。</li>
		<li>在每个样品中加入 1 μL 的 20 毫克/mL 糖原，然后沉淀 1 mL 的冰冷 100% 乙醇（储存在 -20 °C 冰柜中）。短暂混合，然后在-80°C冰柜中孵育样品30分钟至1小时。离心机样品在18，400 x g，10分钟在4°C，并小心地倒出100%乙醇。</li>
		<li>用 500μL 冰冷 70% 乙醇清洗颗粒（储存在 -20 °C 冰柜中）。离心机在18，400 x g 5分钟在4°C。 小心地倒出 70% 的乙醇。</li>
		<li>在 50 °C 下在 1.5 mL 管中孵化样品，直到剩余的乙醇蒸发。在 15μL 的自动克隆 ddH2O 或无核素 ddH 2 O中重新悬浮 DNA 颗粒。</li>
		<li>在120-180 V的1.5%的阿甘蔗凝胶中运行提取的DNA样本，以及DNA阶梯，并检查声波DNA的大小。</li>
	</ol>
</li>
</ol>5. 带珠子的抗体孵育
注：此协议中的以下步骤是大约 2 x 107 神经元细胞与小鼠 ES 细胞分化。不要在 ChIP-exo 协议期间的任何时间冻结和解冻磁珠，因为珠子可能会破裂，导致样品污染，否则抗体的性能可能会受到影响。
<ol><li>细胞裂解和声波化后，为ChIP准备蛋白质G磁珠（材料表），混合磁珠直到均匀，然后将25μL的磁珠加入2 mL蛋白低粘合管中。 注：使用的磁珠类型将取决于抗体的种类。</li>
	<li>用1mL的阻塞液（表2）清洗珠子，混合好，然后放在磁架上1分钟。当仍然在磁架上时，一旦透明，请取出超强剂。</li>
	<li>向磁珠添加 1 mL 的阻塞溶液。在4°C的摇动平台上，在4°C下进行10分钟的岩石管。短暂旋转，然后将管子放在磁架上并去除超强剂。</li>
	<li>重复步骤5.3两次以上。</li>
	<li>向磁珠添加 500μL 的阻塞溶液。短暂地旋转抗体对Isl1（0.04 μg/μL， 材料表），然后添加4μg的抗体到相应的2 mL蛋白低粘合管含有磁珠在阻塞溶液。</li>
	<li>重复步骤 5.5 没有抗体 （即 Chip 的无抗体控制）。 注：通过考虑抗体的质量和用于ChIP的细胞数量，可以经验地确定添加抗体的数量。</li>
	<li>在 4 °C 下的岩石样品，在摇动平台上为 6~24 小时。</li>
</ol>6. 色素免疫沉淀（ChIP）
<ol><li>用 1 mL 的阻塞溶液从步骤 5.8 中清洗抗体涂层珠子。在 4 °C 的摇动平台上放置样品 5 分钟。去除超纳坦。</li>
	<li>再重复步骤 6.1 两次。</li>
	<li>在 50 μL 的阻塞溶液中重新悬索抗体涂层珠子，然后转移到新的 2 mL 蛋白质低粘结管。对于每个 ChIP 样本，在 2 mL 蛋白低粘合管中将声波化的 lysates（从步骤 3.6 到步骤 1.0 mL）添加到抗体涂层珠子中。</li>
	<li>在 4 °C 的摇动平台上将每个样品孵化过夜。</li>
</ol>7. 奇普洗涤
注意：将样品保存在冰上或4°C，以保持ChIP洗涤过程中的蛋白质-DNA交叉链接。
<ol><li>制作高盐洗涤缓冲器、LiCl洗涤缓冲器和 10 mM 三轮车-HCl 缓冲区 （pH 7.4）， 如 表 3中所述。在4°C下存放在50 mL管中。 在使用前将 1000 倍 CPI 库存的 50 μL 添加到所有缓冲区。</li>
	<li>在2 mL蛋白低粘合管中短暂旋转样品，从帽中收集液体，然后放在磁架上1分钟，并用移液器小心地去除超细。</li>
	<li>对于 ChIP 洗涤，在每个管子中添加 1 mL 的裂解缓冲区 3（在 4 °C）。在4°C的摇动平台上混合5分钟。短暂旋转样品，然后放在磁架上 1 分钟，然后用移液器取出超纳坦。</li>
	<li>在每个管子中加入 1 mL 的冷高盐洗涤缓冲器。在4°C的摇动平台上混合5分钟。短暂旋转样品，然后放在磁架上 1 分钟，然后用移液器取出超纳坦。</li>
	<li>在每个管子中加入 1 mL 的冷 LiCl 洗涤缓冲液。在4°C的摇动平台上混合5分钟。短暂旋转样品，然后放在磁架上 1 分钟，然后用移液器取出超纳坦。</li>
	<li>在每个管子中添加 1 mL 的冷 10 mm 三轮车缓冲区 （pH 7.4）。在4°C的摇动平台上混合5分钟。短暂旋转样品，然后放在磁架上 1 分钟，然后用移液器取出超纳坦。 注：可以使用特里斯-EDTA缓冲区（pH 8.0）代替特里斯-HCl缓冲区（pH 7.4）。</li>
	<li>重复步骤 7.4-7.6 三次。</li>
	<li>将带有 500μL 的 Tris-HCl 缓冲区 （pH 7.4） 的样本珠转移到新鲜的 1.5 mL 管中。短暂旋转样品，然后放在磁架上 1 分钟，然后用移液器取出超纳坦。</li>
</ol>8. 结束珠子的修复和尾部反应
注意：声波通常会生成非模糊结束的dsDNA。在 dA 尾随反应之前，需要进行最终修复反应，然后进行钝端 DNA，然后是索引适配器结扎步骤。对于与索引适配器DNA粘性端DNA结扎，dATP通过dA尾部反应添加到钝质dsDNA片段的3'端。末端准备反应组合包含dATP。
<ol><li>ChIP 清洗后，在 1.5 mL 管中的样本珠中加入 38 μL 的自动夹式 ddH2O，用于末端维修和 dA 尾部反应。</li>
	<li>向每个样品添加 5.6 μL 的末端准备反应混合和 2.4 μL 的末端预制酶混合（材料表）（总反应量：46 μL）。在20°C下孵化样品30分钟。</li>
	<li>洗珠如以前的 ChIP 洗涤步骤 7.4+7.6 所述。</li>
</ol>9.珠子上的索引适配器
注：索引适配器具有 6+10 条码索引序列的基础，这些碱基特定于用于高通量测序中多个样本的给定样本。索引适配器DNA序列在 表1中描述。
<ol><li>对于索引适配器连带，在样本珠子中添加 27 μL 的冷 10 m 三叉-HCL 缓冲区 （pH 7.4）。在每个样品中添加 2 μL 的 15 μM 指数适配器、0.5 μL 的韧带增强剂和 15 μL 的韧带主混合（材料表）（总反应量：44.5 μL）。</li>
	<li>在20°C下孵化样品15分钟。洗珠，如步骤 7.4+7.6 所述。</li>
</ol>10. 珠子上的填充反应
注：适配器结对后，适配器的 5'端和 ChIP DNA 的 3' 端之间没有磷酸盐键。刻痕可以通过填充反应修复。
<ol><li>在样品珠中添加 47 μL 的冷 10 m m Tris-HCl 缓冲区 （pH 7.4）。</li>
	<li>在冰上做填充反应混合物（表4 和 材料表）。</li>
	<li>向每个样品添加 11.1 μL 的填充混合物（总反应量：58.1 μL）。在30°C下孵化样品20分钟。洗珠，如步骤 7.4+7.6 所述。</li>
</ol>11. 珠子上的兰姆达外核酶消化
<ol><li>在珠子上填充反应后，在样品珠子中加入 50 μL 的冷高压 ddH2O。</li>
	<li>要在 5' 到 3' 方向消化 ChIP DNA，添加 6 μL 的 10 倍兰姆达外核酶缓冲液和 2 μL 的 5 U/μL 兰姆达外核酶（材料表，总反应量：58 μL）。在37°C下孵化样品30分钟。洗珠，如步骤 7.4+7.6 所述。</li>
</ol>12. 埃卢特和反向交叉链接
<ol><li>制作 表 5中描述的 ChIP 精英缓冲区。 注：不要将CPI添加到ChIP精英缓冲器中。</li>
	<li>要从珠子中分离ChIP样本，请将样品重新悬生在75μL的ChIP排出缓冲液中，并在65°C下孵育15分钟，在130 x g。</li>
	<li>在样品中加入2.5微升20毫克/mL蛋白酶K（材料表）。短暂漩涡，然后在65°C下将样品孵育过夜。</li>
</ol>13. DNA提取
<ol><li>样品在 65 °C 下潜伏过夜后，短暂旋转样品，放在磁架上 1 分钟，然后将每个样品中的超纳坦转移到新的 1.5 mL 管中。</li>
	<li>在样品中添加 1 μL 的 10 毫克/mL RNase A。短暂漩涡，然后在37°C下孵育样品30分钟。添加约25μL的自动夹ddH2O，音量可达100μL。</li>
	<li>使用磁珠（材料表）净化DNA。带 16μL 自动切割 ddH2O 或无核素 ddH2O 的 Elute DNA。</li>
</ol>14. 否认、引漆退化和引漆扩展
<ol><li>在 ChIP 分离和反向交叉链接后，将提取的 DNA 样本的 16 μL 从步骤 13.3 转移到 PCR 管。</li>
	<li>在冰上制作去纳和底漆退化混合物（表6 和 材料表）。向每个样品添加 1.2 μL 的去纳和入门退化混合物（总反应量：17.2 μL）。使用 表 6 中描述的程序运行样本，以对模板 DNA 进行自然和退影入门。</li>
	<li>在冰上制作底漆扩展组合（表7 和 材料表）。</li>
	<li>一旦去自然和安妮素程序完成，在样品中添加3 μL的引漆扩展组合（总反应量：20.2μL）。使用表 7中描述的入门扩展程序运行示例。</li>
	<li>立即进行 dA 尾随反应。</li>
</ol>15. 尾部反应
注：对于粘性端DNA与通用适配器DNA的结合，dATP被添加到钝端的3'端，dA尾部反应。
<ol><li>在冰上制作 dA 尾部混合（表 8 和 材料表）。</li>
	<li>向每个样品添加 4.1 μL 的 dA 尾部混合（总反应量：24.3 μL）。使用程序运行样品进行 dA 尾随（表 8）。</li>
	<li>立即进行通用适配器接合。</li>
</ol>16. 通用适配器连带
注：通用适配器包括用于测序化学DNA样本识别的高通量测序特定序列。通用适配器DNA序列在 表1中描述。
<ol><li>dA尾部反应后，使通用适配器连扎组合（表9 和 材料表）为通用适配器连扎。</li>
	<li>在每个样品中添加 21.5 μL（总反应量：45.8 μL），并在 20 °C 下孵育样品 15 分钟。</li>
</ol>17. DNA清理
<ol><li>使用磁珠（材料表）净化DNA。带 21μL 自动切割 ddh2O 或无核素 ddh2O 的精英 Dna 。</li>
	<li>将样品存放在 -20 °C，直到准备好执行结对介质 PCR （LM-PCR） 和 PCR 净化。</li>
</ol>18. 以利格调解的PCR
注：LM-PCR 引物序列在表 1中描述。
<ol><li>DNA清理后，在冰上进行LM-PCR混合（表10 和 材料表）。</li>
	<li>在每个样品中添加 29 μL 的 LM-PCR 混合（总反应量：50 μL），然后使用 LM-PCR 程序运行样品（表 10）。</li>
	<li>立即进行PCR放大DNA的凝胶净化，然后进行DNA净化，进行高通量测序。</li>
</ol>19.LM-PCR扩音DNA的DNA小便
<ol><li>运行LM-PCR放大DNA样本，以及DNA阶梯，在1.5%的阿加罗斯凝胶在120-180 V和消费税200-400 bp带。</li>
	<li>使用凝胶提取套件（材料表）从去除的凝胶中净化DNA。</li>
	<li>DNA纯化后，检查每个ChIP-exo库样本的浓度（材料表）。提交高通量测序样品，进行单读测序。 注：单读测序的首选读取长度至少为 35 bp，这足以将测序读数与小鼠或人类细胞中的参考基因组进行唯一对齐。对于小鼠或人类细胞中的大多数转录因子，每个 ChIP-exo 样本的读数为 1000 万至 2000 万次，足以识别其基因组结合位置。绘制哺乳动物细胞中富含层石标记的基因组区域图谱需要每个样本至少3000万次读取。</li>
</ol>

<ol>
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作者没有什么可透露的。

在此协议中，ChIP随后的外核酶消化用于获取DNA库，以超高映射分辨率识别哺乳动物细胞中的蛋白质-DNA相互作用。许多变量有助于ChIP-exo实验的质量。关键的实验参数包括抗体的质量、声波的优化以及LM-PCR周期的数量。这些关键的实验参数也是可以限制ChIP-exo实验，并将在下面讨论。
在任何 ChIP 协议中，抗体质量都是最重要的考虑因素之一。建议使用ChIP级抗体。在执行 ChIP-exo ...

蛋白质与DNA相互作用的位置提供了对基因调控机制的洞察。色素免疫沉淀加上高通量测序（ChIP-seq）已经使用了十年来研究活细胞1、2的全基因组蛋白质-DNA相互作用。然而，ChIP-seq方法受DNA片段的异质性和未绑定的DNA污染的限制，导致低映射分辨率、误报、未接听电话和非特定背景信号。ChIP与外核酶消化（ChIP-exo）的结合，通过将ChIPDNA修剪到蛋白质-DNA交叉连接点，提供接近碱基对分辨率和低背景信号3、4、5，从而改进了ChIP-seq方法。ChIP-exo 提供的大大改进的映射分辨率和低背景使整个基因组中能够确定准确和全面的蛋白质-DNA 结合位置。ChIP-exo 能够揭示功能上独特的 DNA 结合图案、转录因子 （TF） 和特定基因组结合位置的多个蛋白质-DNA 交叉链接位点之间的协同相互作用，其他基因组映射方法3、4、6、7无法检测。
ChIP-exo最初用于初露头角的酵母，以检查TF的序列特异性DNA结合，研究转录启动前复合物的精确组织，以及整个基因组4、8、9的单个组酮的亚核体结构。自2011年推出以来，ChIP-exo已成功地用于许多其他生物体，包括细菌、小鼠和人类细胞7、10、11、12、13、14、15、16、17。2016 年，Rhee 等人14首次在哺乳动物神经元中使用 ChIP-exo 来了解在编程 TF Lhx3 降低调节后神经元基因表达是如何维持的，该编程与另一个编程 TF Isl1 形成了一个异质复合体。这项研究表明，在没有Lhx3的情况下，Isl1被招募到受Onecut1 TF约束的新神经元增强剂中，以维持神经元效应基因的基因表达。在这项研究中，ChIP-exo揭示了多个TF如何以接近核苷酸制图分辨率的组合方式动态识别细胞类型和细胞阶段特异性DNA调控元素。其他研究还使用ChIP-exo方法来理解其他哺乳动物细胞系中蛋白质和DNA之间的相互作用。Han等人利用ChIP-exo使用ChIP-exo来研究小鼠红细胞中GATA1和TAL1 TF的全基因组组织。这项研究发现，TAL1是直接招募到DNA，而不是间接通过蛋白质-蛋白质相互作用与GATA1在整个红细胞分化。最近的研究还利用ChIP-exo对CTCF、RNA聚合酶II和平石标记的全基因组结合位置进行剖析，以研究人类细胞系18、19的表观和转录机制。
有几个版本的ChIP-exo协议可用3，5，20。然而，对于不熟悉下一代测序库准备的研究，这些 ChIP-exo 协议很难遵循。ChIP-exo 协议的优秀版本发布时附有易于遵循的说明和视频21，但包含许多酶步骤，需要大量时间才能完成。在这里，我们报告了包含减少酶步骤和潜伏时间的ChIP-exo协议的新版本，以及每个酶步骤21的解释。使用末端预制酶，将末端修复和 dA 尾部反应一步一步地组合在一起。使用夹带增强剂将索引和通用适配器连体步骤的潜伏时间从 2 小时缩短到 15 分钟。删除上一个 ChIP-exo 协议中描述的索引适配器连结步骤后的激酶反应。相反，在寡头DNA合成过程中，磷酸盐组被添加到索引适配器（表1）的5'端之一，这将用于羊田外核酶消化步骤。虽然以前的 ChIP-exo 协议使用 RecJf外核酶消化来消除单链 DNA 污染物，但此消化步骤在这里被删除，因为它对 ChIP-exo 库的质量不至关重要。此外，为了在ChIP分离后净化反向交叉DNA，使用磁珠净化方法代替苯酚：氯仿：异酰醇（PCIA）提取方法。这缩短了DNA提取的潜伏时间。重要的是，它去除索引适配器结对过程中形成的大多数适配器调色器，这可能会影响结对介质 PCR 的效率。
此处提出的ChIP-exo协议进行了优化，用于检测与小鼠胚胎干细胞（ES）细胞分化的哺乳动物神经元的精确蛋白质-DNA相互作用。简言之，收获和交叉链接的神经元细胞被裂解，让色素暴露在声波中，然后进行声波化，从而获得适当大小的DNA片段（图1）。然后，抗体涂层珠子用于有选择地免疫沉淀支离破碎的可溶性色素到感兴趣的蛋白质。当免疫沉淀DNA仍在珠子上时，进行末修复、测序适配器的结对、填充反应和5'至3'lambda外核酶消化步骤。外核酶消化步骤使ChIP-exo具有超高分辨率和高信号噪声比。Lambda 外核酶将免疫沉淀的 DNA 从交叉连接部位修剪出几个碱基对 （bp），从而导致污染的 DNA 降解。外核酶处理的ChIP DNA从抗体涂层珠子中分离出，蛋白质-DNA交叉链路被逆转，蛋白质退化。脱氧核糖核酸被提取并去自然到单链ChIPDNA，然后引物退化和扩展，使双链DNA（dsDNA）。接下来，执行将通用适配器与外核酶处理末端的连结。由此产生的DNA被纯化，然后PCR放大，凝胶纯化，并接受下一代测序。
ChIP-exo 协议比 ChIP-seq 协议长，但在技术上并不十分具有挑战性。任何成功的免疫沉淀的ChIPDNA都可以接受ChIP-exo，并具有几个额外的酶步骤。ChIP-exo在基因组结合位点方面的显著优势，如超高映射分辨率、减少背景信号、减少误报和阴性，超过了时间成本。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Agarose, UltraPure</td>
			<td >Invitrogen</td>
			<td >16500</td>
			<td >Checking Sonication (Section 4.3.6) and Gel Purification of LM-PCR Amplified DNA (Section 19.1)</td>
		</tr>
		<tr >
			<td >Albumin, Bovine Serum (BSA), Protease Free, Heat Shock Isolation, Min. 98%</td>
			<td >BioShop</td>
			<td >ALB003</td>
			<td >Blocking Solution</td>
		</tr>
		<tr >
			<td >Antibody against Isl1</td>
			<td >DSHB</td>
			<td >39.3F7</td>
			<td >Antibody incuation with beads (Section 5.6)</td>
		</tr>
		<tr >
			<td >Bioruptor Pico&#160;</td>
			<td >Diagenode</td>
			<td >B01060010</td>
			<td >Sonicating Chromatin (Section 3.3)</td>
		</tr>
		<tr >
			<td >Bovine serum albumin (BSA), Molecular Biology Grade</td>
			<td >New England BioLabs</td>
			<td >B9000S</td>
			<td >Fill-in Reaction on Beads (Section 10.2) and Denaturing, Primer Annealing and Primer Extension (Section 14.2)</td>
		</tr>
		<tr >
			<td >Centrifuge 5424 R</td>
			<td >Eppendorf</td>
			<td >5404000138</td>
			<td >Sonicating Chromatin (Section 3.5), Checking Sonication (Section 4.3.1, 4.3.3 and 4.3.4)</td>
		</tr>
		<tr >
			<td >Centrifuge 5804 R</td>
			<td >Eppendorf</td>
			<td >22623508</td>
			<td >Harvest, cross-linking and freezing cells (Section 1.3), Cell lysis (Section 2.2 and 2.3), Sonicating Chromatin (Section 3.1)</td>
		</tr>
		<tr >
			<td >cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail</td>
			<td >Roche</td>
			<td >4693159001</td>
			<td >Added to all buffers, except Proteinase K buffer and ChIP Elution buffer</td>
		</tr>
		<tr >
			<td >dATP Solution</td>
			<td >New England BioLabs</td>
			<td >N0440S</td>
			<td >dA-Tailing Reaction (Section 15.1)</td>
		</tr>
		<tr >
			<td >Deoxycholic Acid Sodium Salt</td>
			<td >fisher scientific</td>
			<td >BP349</td>
			<td >Lysis Buffer 3, High Salt Wash Buffer and LiCl Wash Buffer</td>
		</tr>
		<tr >
			<td >dNTP Mix, Molecular Biology Grade</td>
			<td >Thermo Scientific</td>
			<td >R0192</td>
			<td >Fill-in Reaction on Beads (Section 10.2) and Denaturing, Primer Annealing and Primer Extension (Section 14.2)</td>
		</tr>
		<tr >
			<td >DreamTaq Green PCR Master Mix, 2x&#160;</td>
			<td >Thermo Scientific</td>
			<td >K1081</td>
			<td >Ligation-Mediated PCR (Section 18.1)</td>
		</tr>
		<tr >
			<td >EDTA, 0.5 M, Sterile Solution, pH 8.0</td>
			<td >BioShop</td>
			<td >EDT111</td>
			<td >Lysis Buffer 1-3, Checking Sonication (Section 4.1), High Salt Wash Buffer, LiCl Wash Buffer and ChIP Elution Buffer</td>
		</tr>
		<tr >
			<td >Ethyl Alcohol Anhydrous, 100%</td>
			<td >Commercial alcohols</td>
			<td >P006EAAN</td>
			<td >Checking Sonication (Section 4.3.3)</td>
		</tr>
		<tr >
			<td >Formaldehyde, 36.5-38%, contains 10-15% methanol</td>
			<td >Sigma</td>
			<td >F8775</td>
			<td >Harvest, cross-linking and freezing cells (Section 1.1)</td>
		</tr>
		<tr >
			<td >Glycerol, Reagent Grade, min 99.5%</td>
			<td >BioShop</td>
			<td >GLY002</td>
			<td >Lysis Buffer 1</td>
		</tr>
		<tr >
			<td >Glycine, Biotechnology Grade, min. 99%</td>
			<td >BioShop</td>
			<td >GLN001</td>
			<td >Harvest, cross-linking and freezing cells (Section 1.2)</td>
		</tr>
		<tr >
			<td >Glycogen, RNA Grade</td>
			<td >Thermo Scientific</td>
			<td >R0551</td>
			<td >Checking Sonication (Section 4.3.3)</td>
		</tr>
		<tr >
			<td >HEPES, 1 M Sterile-filtered Solution, pH 7.3&#160;</td>
			<td >BioShop</td>
			<td >HEP003</td>
			<td >Lysis Buffer 1, High Salt Wash Buffer</td>
		</tr>
		<tr >
			<td >Klenow Fragment (3-&#62;5 exo-)</td>
			<td >New England BioLabs</td>
			<td >M0212S</td>
			<td >dA-Tailing Reaction (Section 15.1)</td>
		</tr>
		<tr >
			<td >Lambda exonuclease</td>
			<td >New England BioLabs</td>
			<td >M0262S</td>
			<td >Lambda Exonuclease Digestion on Beads (Section 11.2)</td>
		</tr>
		<tr >
			<td >Ligase Enhancer</td>
			<td >New England BioLabs</td>
			<td >E7645S</td>
			<td >NEBNext Ultra II DNA Library Kit. Index Adapter Ligation on Beads (Section 9.1) and Universal Adapter Ligation (Section 16.1)</td>
		</tr>
		<tr >
			<td >Ligase Master Mix</td>
			<td >New England BioLabs</td>
			<td >E7645S</td>
			<td >NEBNext Ultra II DNA Library Kit. Index Adapter Ligation on Beads (Section 9.1) and Universal Adapter Ligation (Section 16.1)</td>
		</tr>
		<tr >
			<td >Lithium Chloride (LiCl), Reagent grade</td>
			<td >Bioshop</td>
			<td >LIT704</td>
			<td >LiCl Wash Buffer</td>
		</tr>
		<tr >
			<td >Magnetic beads for ChIP (Dynabeads Protein G)</td>
			<td >Dynabeads Protein G (magnetic beads for ChIP)</td>
			<td >Dynabeads Protein G (magnetic beads for ChIP)</td>
			<td >Antibody incubation with beads (Section 5)</td>
		</tr>
		<tr >
			<td >Magnetic beads for DNA purification (AMPure XP Beads)</td>
			<td >Beckman Coulter</td>
			<td >A63880</td>
			<td >DNA Extraction (Section 13.3) and DNA Clean-up (Section 17.1)</td>
		</tr>
		<tr >
			<td >Magnetic rack (DynaMag-2 Magnet)</td>
			<td >Invitrogen</td>
			<td >12321D</td>
			<td >Used in many steps in Sections: 5 - 11, 13</td>
		</tr>
		<tr >
			<td >MinElute Gel Extraction Kit</td>
			<td >Qiagen&#160;</td>
			<td >28604</td>
			<td >Gel Purification of PCR Amplified DNA (Section 19.2)</td>
		</tr>
		<tr >
			<td >N-Lauroylsarcosine sodium salt solution, 30% aqueous solution, &#8805;97.0% (HPLC)</td>
			<td >Sigma</td>
			<td >61747</td>
			<td >Lysis Buffer 3</td>
		</tr>
		<tr >
			<td >Octylphenol Ethoxylate (IGEPAL CA630)</td>
			<td >BioShop</td>
			<td >NON999</td>
			<td >Lysis Buffer 1 and LiCl Wash Buffer</td>
		</tr>
		<tr >
			<td >Phenol:Chloroform:Isoamyl Alcohol, Biotechnology Grade (25:24:1)</td>
			<td >BioShop</td>
			<td >PHE512</td>
			<td >Checking Sonication (Section 4.3.1)</td>
		</tr>
		<tr >
			<td >phi29 DNA Polymerase</td>
			<td >New England BioLabs</td>
			<td >M0269L</td>
			<td >Fill-in Reaction on Beads (Section 10.2) and Denaturing, Primer Annealing and Primer Extension (Section 14.3 and 14.4)</td>
		</tr>
		<tr >
			<td >Phosphate-Buffered Saline (PBS), 1x&#160;</td>
			<td >Corning</td>
			<td >21040CV</td>
			<td >Harvest, cross-linking and freezing cells (Section 1.3) and Sonicating Chromatin (Section 3.1), Antibody incuation with beads (Section 5.1)</td>
		</tr>
		<tr >
			<td >PowerPac Basic Power Supply</td>
			<td >BioRad</td>
			<td >1645050</td>
			<td >Checking Sonication (Section 4.3.6) and Gel Purification of LM-PCR Amplified DNA (Section 19.1)</td>
		</tr>
		<tr >
			<td >ProFlex PCR System</td>
			<td >Applied Biosystems</td>
			<td >ProFlex PCR System</td>
			<td >Used in Sections: 14.2, 14.4, 15.2, 16.2 and 18.2</td>
		</tr>
		<tr >
			<td >Protein LoBind Tube, 2.0 mL&#160;</td>
			<td >Eppendorf</td>
			<td >22431102</td>
			<td >Antibody Incubation with Beads (Section 5.2) and Chromatin Immunoprecipitation (Section 6.3)</td>
		</tr>
		<tr >
			<td >Proteinase K Solution, RNA Grade</td>
			<td >Invitrogen</td>
			<td >25530049</td>
			<td >Checking Sonication (Section 4.2) and Elution and Reverse Crosslinking (Section 12.3)</td>
		</tr>
		<tr >
			<td >Qubit 4.0 Fluorometer</td>
			<td >Invitrogen</td>
			<td >Q33226</td>
			<td >Gel Purification of PCR Amplified DNA (Section 19.3)</td>
		</tr>
		<tr >
			<td >Quibit dsDNA BR assay kit</td>
			<td >Invitrogen</td>
			<td >Q32853</td>
			<td >Gel Purification of PCR Amplified DNA (Section 19.3)</td>
		</tr>
		<tr >
			<td >Rnase A, Dnase and Protease-free</td>
			<td >Thermo Scientific</td>
			<td >EN0531</td>
			<td >Checking Sonication (Section 4.3.2) and DNA Extraction (Section 13.2)</td>
		</tr>
		<tr >
			<td >Sodium chloride (NaCl), BioReagent&#160;</td>
			<td >Sigma</td>
			<td >S5886</td>
			<td >Lysis Buffer 1-3, High Salt Wash Buffer</td>
		</tr>
		<tr >
			<td >Sodium Dodecyl Sulfate (SDS), Electrophoresis Grade</td>
			<td >BioShop</td>
			<td >SDS001</td>
			<td >Checking Sonication (Section 4.1), High Salt Wash Buffer and ChIP Elution Buffer</td>
		</tr>
		<tr >
			<td >Sonication beads and 15 mL Bioruptor Tubes</td>
			<td >Diagenode</td>
			<td >C01020031</td>
			<td >Sonicating Chromatin (Section 3.1 and 3.2)</td>
		</tr>
		<tr >
			<td >ThermoMixer F1.5&#160;</td>
			<td >Eppendorf</td>
			<td >5384000020</td>
			<td >Section 4.2, 4.3.2, 4.3.5, 8.2, 9.2, 10.3, 11.2, 12.2, 12.3, 13.2 and 16.2</td>
		</tr>
		<tr >
			<td >Trizma hydrochloride solution (Tris-HCl), BioPerformance Certified, 1 M, pH 7.4</td>
			<td >Sigma</td>
			<td >T2194</td>
			<td >10 mM Tris-HCl Buffer</td>
		</tr>
		<tr >
			<td >Trizma hydrochloride solution (Tris-HCl), BioPerformance Certified, 1 M, pH 8.0</td>
			<td >Sigma</td>
			<td >T2694</td>
			<td >Lysis Buffer 2, Lysis Buffer 3, Checking Sonication (Section 4.1), LiCl Wash Buffer and ChIP Elution Buffer</td>
		</tr>
		<tr >
			<td >Ultra II End Repair/dA-Tailing Module (24 rxn -&#62; 120 rxn)</td>
			<td >New England BioLabs</td>
			<td >E7546S</td>
			<td >End Prep Reaction mix and End Prep Enzyme mix. End-repair and dA-Tailing Reaction on Beads (Section 8.2)</td>
		</tr>
		<tr >
			<td >VWR Mini Tube Rocker, Variable Speed</td>
			<td >VWR</td>
			<td >10159-752</td>
			<td >Used in many steps in sections: 1, 2, 5, 6 and 7</td>
		</tr>
		<tr >
			<td >2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, Triton X-100, Reagent Grade</td>
			<td >BioShop</td>
			<td >TRX506</td>
			<td >Lysis Buffer 1, Sonicating Chromatin (Section 3.4) and High Salt Wash Buffer</td>
		</tr>
	</tbody>
</table>

high-resolution mapping of protein-dna interactions in mouse stem cell-derived neurons using chromatin immunoprecipitation-exonuclease (chip-exo)

识别基因组上特定的蛋白质-DNA相互作用对于理解基因调控具有重要意义。色素免疫沉淀与高通量测序（ChIP-seq）广泛用于识别DNA结合蛋白的全基因组结合位置。然而，ChIP-seq方法受其声波DNA片段长度和非特定背景DNA的异质性限制，导致DNA结合位点的制图分辨率低且不确定。为了克服这些限制，ChIP与外核酶消化（ChIP-exo）的结合利用5'到3'外核酶消化来修剪异构大小的免疫沉淀DNA到蛋白质-DNA交叉连接部位。外核酶治疗也消除了非特定背景DNA。图书馆准备的和外核酶消化的DNA可以发送用于高通量测序。ChIP-exo 方法允许近基对映射分辨率，具有更高的检测灵敏度和更小的背景信号。下文描述了哺乳动物细胞和下一代测序的优化 ChIP-exo 协议。

图2A说明了从小鼠ES细胞中分离出来的运动神经元细胞的细胞裂解和声波化后产生的声波结果。声波周期的最佳数量（例如图 2A中的12个周期）在100-400 bp DNA片段中产生强烈的DNA强度。高质量的 ChIP-exo 库基于支离破碎的染色质 DNA 的大小和数量。因此，建议在启动ChIP-exo之前，对每个细胞类型和批次细胞进行声波优化。
<strong class="xfig...

Watch this Scientific Journal Video about High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease ChIP-Exo at JoVE.com

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease ChIP-Exo

精确确定整个基因组中的蛋白质结合位置对于理解基因调控非常重要。在这里，我们描述了一种基因组映射方法，它用外核酶消化（ChIP-exo）处理染色质免疫沉淀DNA，然后进行高通量测序。该方法可检测哺乳动物神经元中具有近碱基对映射分辨率和高信号噪声比的蛋白质-DNA相互作用。

使用色素免疫沉淀-外核酶（ChIP-Exo）对小鼠干细胞衍生神经元中的蛋白质-DNA相互作用进行高分辨率映射

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled ...

high-resolution-mapping-protein-dna-interactions-mouse-stem-cell

Research

JoVE Journal

Genetics

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo)

4.7K 次观看.  University of Toronto. 精确确定整个基因组中的蛋白质结合位置对于理解基因调控非常重要。在这里，我们描述了一种基因组映射方法，它用外核酶消化（ChIP-exo）处理染色质免疫沉淀DNA，然后进行高通量测序。该方法可检测哺乳动物神经元中具有近碱基对映射分辨率和高信号噪声比的蛋白质-DNA相互作用。

视频: High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease ChIP-Exo

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

使用生物素化补骨脂素全球定位RNA-RNA相互作用

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

科研

遗传学

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

与ChEC-seq的蛋白质-DNA相互作用的全基因组映射<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational modifications (PTMs) of histone tails, the exchange of canonical histones with histone variants, and nucleosome eviction. Such regulation requires the binding of signal-sensitive transcription factors (TFs) that recruit chromatin-modifying enzymes at regulatory elements defined as enhancers. Understanding how signaling cascades regulate enhancer activity requires a comprehensive analysis of the binding of TFs, chromatin modifying enzymes, and the occupancy of specific histone marks and histone variants. Chromatin immunoprecipitation (ChIP) assays utilize highly specific antibodies to immunoprecipitate specific protein/DNA complexes. The subsequent analysis of the purified DNA allows for the identification the region occupied by the protein recognized by the antibody. This work describes a protocol to efficiently perform ChIP of histone proteins in a mature mouse T-cell line. The presented protocol allows for the performance of ChIP assays in a reasonable timeframe and with high reproducibility.

Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines

This work describes a protocol for chromatin immunoprecipitation (ChIP) using a mature mouse T-cell line. This protocol is suitable to investigate the distribution of specific histone marks at specific promoter sites or genome-wide.

染色质免疫沉淀（ChIP）在小鼠T细胞系

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational ...

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV integration sites can be used as functional markers of active regulatory elements. Here, we present a retroviral scanning tool, which allows the genome-wide identification of cell-specific enhancers and promoters. Briefly, the target cell population is transduced with an mLV-derived vector and genomic DNA is digested with a frequently cutting restriction enzyme. After ligation of genomic fragments with a compatible DNA linker, linker-mediated polymerase chain reaction (LM-PCR) allows the amplification of the virus-host genome junctions. Massive sequencing of the amplicons is used to define the mLV integration profile genome-wide. Finally, clusters of recurrent integrations are defined to identify cell-specific regulatory regions, responsible for the activation of cell-type specific transcriptional programs.
The retroviral scanning tool allows the genome-wide identification of cell-specific promoters and enhancers in prospectively isolated target cell populations. Notably, retroviral scanning represents an instrumental technique for the retrospective identification of rare populations (e.g. somatic stem cells) that lack robust markers for prospective isolation.

Here, we describe a protocol for genome-wide mapping of the integration sites of Moloney murine leukemia virus-based retroviral vectors in human cells.

逆转录病毒扫描：映射MLV集成站点以定义细胞特异性监管区域

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

高分辨率荧光原位杂交在果蝇胚胎和组织中使用 Tyramide 信号放大

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions of chromatin. These regions represent regulatory DNA elements (e.g., promoters, enhancers, locus control regions, insulators) to which transcription factors bind. Mapping the accessible chromatin landscape is a powerful approach for uncovering active regulatory elements across the genome. This information serves as an unbiased approach for discovering the network of relevant transcription factors and mechanisms of chromatin structure that govern gene expression programs. ATAC-seq is a robust and sensitive alternative to DNase I hypersensitivity analysis coupled with next-generation sequencing (DNase-seq) and formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) for genome-wide analysis of chromatin accessibility and to the sequencing of micrococcal nuclease-sensitive sites (MNase-seq) to determine nucleosome positioning. We present a detailed ATAC-seq protocol optimized for human primary immune cells i.e. CD4+ lymphocytes (T helper 1 (Th1) and Th2 cells). This comprehensive protocol begins with cell harvest, then describes the molecular procedure of chromatin tagmentation, sample preparation for next-generation sequencing, and also includes methods and considerations for the computational analyses used to interpret the results. Moreover, to save time and money, we introduced quality control measures to assess the ATAC-seq library prior to sequencing. Importantly, the principles presented in this protocol allow its adaptation to other human immune and non-immune primary cells and cell lines. These guidelines will also be useful for laboratories which are not proficient with next-generation sequencing methods.

Assay for Transposase-Accessible Chromatin coupled with high-throughput sequencing (ATAC-seq) is a genome-wide method to uncover accessible chromatin. This is a step-by-step ATAC-seq protocol, from molecular to the final computational analysis, optimized for human lymphocytes (Th1/Th2). This protocol can be adopted by researchers without prior experience in next-generation sequencing methods.

ATAC 基因组在原发性人类 T 淋巴细胞中的染色体全易接近染色质

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions ...

Promoter Capture Hi-C: High-resolution, Genome-wide Profiling of Promoter Interactions

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the spatio-temporal expression of their target genes through physical contact, often bridging considerable (in some cases hundreds of kilobases) genomic distances and bypassing nearby genes. The human genome harbors an estimated one million enhancers, the vast majority of which have unknown gene targets. Assigning distal regulatory regions to their target genes is thus crucial to understand gene expression control. We developed Promoter Capture Hi-C (PCHi-C) to enable the genome-wide detection of distal promoter-interacting regions (PIRs), for all promoters in a single experiment. In PCHi-C, highly complex Hi-C libraries are specifically enriched for promoter sequences through in-solution hybrid selection with thousands of biotinylated RNA baits complementary to the ends of all promoter-containing restriction fragments. The aim is to then pull-down promoter sequences and their frequent interaction partners such as enhancers and other potential regulatory elements. After high-throughput paired-end sequencing, a statistical test is applied to each promoter-ligated restriction fragment to identify significant PIRs at the restriction fragment level. We have used PCHi-C to generate an atlas of long-range promoter interactions in dozens of human and mouse cell types. These promoter interactome maps have contributed to a greater understanding of mammalian gene expression control by assigning putative regulatory regions to their target genes and revealing preferential spatial promoter-promoter interaction networks. This information also has high relevance to understanding human genetic disease and the identification of potential disease genes, by linking non-coding disease-associated sequence variants in or near control sequences to their target genes.

DNA regulatory elements, such as enhancers, control gene expression by physically contacting target gene promoters, often through long-range chromosomal interactions spanning large genomic distances. Promoter Capture Hi-C (PCHi-C) identifies significant interactions between promoters and distal regions, enabling the assignment of potential regulatory sequences to their target genes.

启动子捕获高分辨率, 全基因组的启动子交互分析

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

CRISPR 的染色质环结构重组

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

High-Resolution Comparison of Bacterial Conjugation Frequencies

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons of conjugation frequency under different conditions are required to understand how the conjugative element spreads in nature. However, conventional methods for comparing conjugation frequency are not appropriate for in-depth comparisons because of the high background caused by the occurrence of additional conjugation events on the selective plate. We successfully reduced the background by introducing a most probable number (MPN) method and a higher concentration of antibiotics to prevent further conjugation in selective liquid medium. In addition, we developed a protocol for estimating the probability of how often donor cells initiate conjugation by sorting single donor cells into recipient pools by fluorescence-activated cell sorting (FACS). Using two plasmids, pBP136 and pCAR1, the differences in conjugation frequency in Pseudomonas putida cells could be detected in liquid medium at different stirring rates. The frequencies of conjugation initiation were higher for pBP136 than for pCAR1. Using these results, we can better understand the conjugation features in these two plasmids.

With an aim to understand the behaviors of various bacterial conjugative DNA elements under different conditions, we describe a protocol for detecting differences in conjugation frequency, with high resolution, to estimate how efficiently the donor bacterium initiates conjugation.

细菌结合频率的高分辨率比较

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons ...

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene expression. The cellular transcriptional machinery and its chromatin modification proteins coordinate to regulate transcription. To analyze transcriptional regulation at the molecular level, several experimental methods such as electrophoretic mobility shift, transient reporter and chromatin immunoprecipitation (ChIP) assays are available. We describe a modified ChIP assay in detail in this article because of its advantages in directly showing histone modifications and the interactions between proteins and DNA in cells. One of the key steps in a successful ChIP assay is chromatin shearing. Although sonication is commonly used for shearing chromatin, it is difficult to identify reproducible conditions. Instead of shearing chromatin by sonication, we utilized enzymatic digestion with micrococcal nuclease (MNase) to obtain more reproducible results. In this article, we provide a straightforward ChIP assay protocol using MNase.

Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.

使用哺乳动物细胞中的微球菌核酸酶进行染色质免疫沉淀测定

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...