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><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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>BioShop</td><td>HEP003</td><td>Lysis Buffer 1, High Salt Wash Buffer</td></tr><tr><td>Klenow Fragment (3-&gt;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&amp;nbsp;</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, &amp;ge;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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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 -&gt; 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。
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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

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 ...

科研

遗传学

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 ...

Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory regions of oncogenes and tumor suppressors can lead to changes in gene expression and play an important role in the pathogenesis of brain tumors. Native chromatin immunoprecipitation (ChIP) is a popular technique that allows the detection of modifications or other proteins tightly bound to DNA. In contrast to cross-linked ChIP, in native ChIP, cells are not treated with formaldehyde to covalently link protein to DNA. This is advantageous because sometimes crosslinking may fix proteins that only transiently interact with DNA and do not have functional significance in gene regulation. In addition, antibodies are generally raised against unfixed peptides. Therefore, antibody specificity is increased in native ChIP. However, it is important to keep in mind that native ChIP is only applicable to study histones or other proteins that bind tightly to DNA. This protocol describes the native chromatin immunoprecipitation on murine brain tumor neurospheres.

Epigenetic mechanisms are frequently altered in glioma. Chromatin immunoprecipitation could be used to study the consequences of genetic alterations in glioma that result from changes in histone modifications which regulate chromatin structure and gene transcription. This protocol describes native chromatin immunoprecipitation on murine brain tumor neurospheres.

利用小鼠脑肿瘤球的原发染色质沉淀

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory ...

癌症研究

A Method to Study de novo Formation of Chromatin Domains

The organization and structure of chromatin domains are unique to individual cell lineages. Their misregulation might lead to a loss in cellular identity and/or disease. Despite tremendous efforts, our understanding of the formation and propagation of chromatin domains is still limited. Chromatin domains have been studied under steady-state conditions, which are not conducive to following the initial events during their establishment. Here, we present a method to inducibly reconstruct chromatin domains and follow their re-formation as a function of time. Although, first applied to the case of PRC2-mediated repressive chromatin domain formation, it could be easily adapted to other chromatin domains. The modification of and/or the combination of this method with genomics and imaging technologies will provide invaluable tools to study the establishment of chromatin domains in great detail. We believe that this method will revolutionize our understanding of how chromatin domains form and interact with each other.

A Method to Study de novo Formation of Chromatin Domains

This method is designed to follow formation of PRC2-mediated chromatin domains in cell lines, and the method can be adapted to many other systems.

色质域新形成研究方法

The organization and structure of chromatin domains are unique to individual cell lineages. Their misregulation might lead to a loss in cellular identity ...

CARIP-Seq and ChIP-Seq: Methods to Identify Chromatin-Associated RNAs and Protein-DNA Interactions in Embryonic Stem Cells

Embryonic stem (ES) cell self-renewal&#160;and differentiation is governed by extrinsic signals and intrinsic networks of transcription factors, epigenetic regulators, and post-translation modifications of histones that combinatorially influence the gene expression state of nearby genes. RNA has also been shown to interact with various proteins to regulate chromatin dynamics&#160;and gene expression. Chromatin-associated RNA immunoprecipitation (CARIP) followed by next-generation sequencing (CARIP-Seq) is a novel method to survey RNAs associated with chromatin proteins, while chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq) is a powerful genomics technique to map the location of post-translational modification of histones, transcription factors, and epigenetic modifiers on a global-scale in ES cells. Here, we describe methods to perform CARIP-Seq and ChIP-Seq, including library construction for next-generation sequencing, to generate global chromatin-associated RNA and epigenomic maps in ES cells.

Here, we describe methods to perform ChIP-Seq and CARIP-Seq, including library preparation for next-generation sequencing, to generate global epigenomic and chromatin-associated RNA maps in ES cells.

CARIP 和芯片序列: 鉴定胚胎干细胞中染色质相关 rna 和蛋白质-DNA 相互作用的方法

Embryonic stem (ES) cell self-renewal&#160;and differentiation is governed by extrinsic signals and intrinsic networks of transcription factors, ...

发育生物学

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. Additionally, epigenetic chromatin modifications, like histone methylation, have an important role for establishing and maintaining specific cell fates within this process. The vast majority of histone methylation occurs on the flexible histone tail, which is accessible to histone modifiers, erasers, and histone reader proteins. In contrast, H3K79 methylation is located in the globular domain of histone 3 and is implicated in different developmental functions. H3K79 methylation is evolutionarily conserved and can be found in a wide range of species from Homo sapiens to Saccharomyces cerevisiae. The modification occurs in different cell populations within organisms, including neural progenitors. The location of H3K79 methylation in the globular domain of histone 3 makes it difficult to assess. Here, we present methods to isolate and culture cortical progenitor cells (CPCs) from embryonic cortical brain tissue (E11.5-E14.5) or cerebellar granular neuron progenitors (CGNPs) from postnatal tissue (P5-P7), and to efficiently immunoprecipitate H3K79me2 for quantitative PCR (qPCR) and genome-wide sequencing.

We present an effective and reproducible method to isolate and culture neural progenitor cells from embryonic and postnatal brain tissue for chromatin immunoprecipitation (ChIP) of histone 3 lysine 79 dimethylation (H3K79me2) - a histone mark located within the globular domain of histone 3.

组蛋白3赖氨酸 79 Dimethylation 标记沉淀后神经祖细胞的分离培养

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. ...

神经科学

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

代高通量测序技术（ChIP-seq的高品质的染色质免疫沉淀DNA模板）

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

生物学

The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision

Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA interactions. ChIP coupled to high throughput sequencing (ChIP-seq) is commonly used to determine the genomic location of proteins that interact with chromatin. However, ChIP-seq is hampered by relatively low mapping resolution of several hundred base pairs and high background signal. The ChIP-exo method is a refined version of ChIP-seq that substantially improves upon both resolution and noise. The key distinction of the ChIP-exo methodology is the incorporation of lambda exonuclease digestion in the library preparation workflow to effectively footprint the left and right 5&#39; DNA borders of the protein-DNA crosslink site. The ChIP-exo libraries are then subjected to high throughput sequencing. The resulting data can be leveraged to provide unique and ultra-high resolution insights into the functional organization of the genome. Here, we describe the ChIP-exo method that we have optimized and streamlined for mammalian systems and next-generation sequencing-by-synthesis platform.

Here, we present a protocol to achieve near base pair resolution of protein-DNA interactions. This is obtained by exonuclease treatment of DNA fragments selectively enriched by chromatin immunoprecipitation (ChIP-exo) followed by high throughput sequencing.

该芯片外型方法:确定蛋白质-DNA相互作用与近基部精密配对

Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

tchip-seq: 细胞专用表观体分析

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...

Deciphering High-Resolution 3D Chromatin Organization via Capture Hi-C

The spatial organization of the genome contributes to its function and regulation in many contexts, including transcription, replication, recombination, and repair. Understanding the exact causality between genome topology and function is therefore crucial and increasingly the subject of intensive research. Chromosome conformation capture technologies (3C) allow inferring the 3D structure of chromatin by measuring the frequency of interactions between any region of the genome. Here we describe a fast and simple protocol to perform Capture Hi-C, a 3C-based target enrichment method that characterizes the allele-specific 3D organization of megabased-sized genomic targets at high-resolution. In Capture Hi-C, target regions are captured by an array of biotinylated probes before downstream high-throughput sequencing. Thus, higher resolution and allele-specificity are achieved while improving the time-effectiveness and affordability of the technology. To demonstrate its strengths, the Capture Hi-C protocol was applied to the mouse X-inactivation center (Xic), the master regulatory locus of X-chromosome inactivation (XCI).

Deciphering High-Resolution 3D Chromatin Organization via Capture Hi-C

This protocol describes the Capture Hi-C method used to characterize the 3D organization of megabased-sized targeted genomic regions at high-resolution, including boundaries of topologically associating domains (TADs) and long-range chromatin interactions between regulatory and other DNA sequence elements.

通过捕获Hi-C 破 译高分辨率3D染色质组织

The spatial organization of the genome contributes to its function and regulation in many contexts, including transcription, replication, recombination, ...

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

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与ChEC-seq的蛋白质-DNA相互作用的全基因组映射

染色质免疫沉淀（ChIP）在小鼠T细胞系

利用小鼠脑肿瘤球的原发染色质沉淀

色质域新形成研究方法

CARIP 和芯片序列: 鉴定胚胎干细胞中染色质相关 rna 和蛋白质-DNA 相互作用的方法

组蛋白3赖氨酸 79 Dimethylation 标记沉淀后神经祖细胞的分离培养

代高通量测序技术（ChIP-seq的高品质的染色质免疫沉淀DNA模板）

该芯片外型方法:确定蛋白质-DNA相互作用与近基部精密配对

tchip-seq: 细胞专用表观体分析

通过捕获Hi-C 破译高分辨率3D染色质组织

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

摘要

探索更多视频

此视频中的章节

与ChEC-seq的蛋白质-DNA相互作用的全基因组映射

染色质免疫沉淀（ChIP）在小鼠T细胞系

利用小鼠脑肿瘤球的原发染色质沉淀

色质域新形成研究方法

CARIP 和芯片序列: 鉴定胚胎干细胞中染色质相关 rna 和蛋白质-DNA 相互作用的方法

组蛋白3赖氨酸 79 Dimethylation 标记沉淀后神经祖细胞的分离培养

代高通量测序技术（ChIP-seq的高品质的染色质免疫沉淀DNA模板）

该芯片外型方法:确定蛋白质-DNA相互作用与近基部精密配对

tchip-seq: 细胞专用表观体分析

通过捕获Hi-C 破 译高分辨率3D染色质组织

通过捕获Hi-C 破译高分辨率3D染色质组织