作者感谢 1月 Appel 在制定本议定书期间提供了出色的技术援助。在拉达实验室的工作是支持 CMMC 内部资助, DFG 研究补助金 (ra 2547/1-1, ra 2547/2-1, 和 TE 1007/3-1), UoC 高级研究员组赠款, 和 CECAD 补助金。

根据德国动物保育指南, 没有机构动物保育和使用委员会 (IACUC) 批准是进行鸡胚实验所必需的。根据当地的指导方针, 只有鸡 HH44 (18 天) 的胚胎和年龄的试验需要 IACUC 批准。然而, 这项研究中使用的胚胎都处于胚胎发育的早期阶段 ( 即, HH19 (72 h)). 
 注意: 本协议的目的是提供芯片分析的详细说明, 以便它可以有效地与 qPCR 或下一代测序 ( 即, 芯片-seq) 结合, 以调查组蛋白修饰丰胚胎样本 (范围从 5 x 10 4 -5 x 10 5 细胞为每个芯片反应) 和不同的组织类型 ( 图 1 )。该协议应适用于调查转录因子和 co-activators ( 例如, p300) 的绑定配置文件。然而, 由于这些调节蛋白的丰度较低, 可能需要较大的细胞数 (5 x 10 5 -5 x 10 6 单元格用于每个芯片反应). 
 1. 鸡蛋的制备和鸡 SNT 的显微切割 注意: microdissections 的过程从组织到组织, 从动物模型到动物模型, 技术上不同。本节详细介绍了用于从 stage-HH19 鸡胚中获取臂 SNT 段的解剖协议. 
<ol> <li> 孵育可育的白色航母鸡卵, 从当地饲养员获得, 在水平方向为37和 #176; C 和80% 湿度3天, 以获得 stage-HH19 鸡胚. </li>
	<li> 根据汉堡包和哈密尔顿鸡胚胎发育分期系统确定阶段 17 . </li>
	<li> 在冷条件下执行所有 microdissections; 否则, 染色质完整性可能会受到影响. </li>
	<li> 隔离 HH19 的胚胎. 
	<ol> <li> 为了使胚胎可访问, 使用10毫升注射器 (0.90 x 40 mm) 提取3毫升的白蛋白, 以降低胚. </li>
		<li> 使用细剪刀在鸡蛋上做一个小开口, 并添加1毫升的洛克解决方案 (参见 补充材料 为食谱), 以方便去除 extra-embryonic 膜. </li>
		<li> 在胚下使用1毫升注射器 (0.55 x 25 mm 2 ) 注入10% 印第安黑墨水/洛克溶液, 使胚胎的可视化更容易. </li>
		<li> 用医用手术用的精细剪刀和镊子切断环绕胚胎的 extra-embryonic 膜. </li>
		<li> 使用穿孔勺将胚胎移植到4.5 厘米的培养皿中, 其中含有20毫升的 1x PBS 溶液. </li>
	</ol> </li> <li> 在显微镜下用细剪刀和镊子解剖 extra-embryonic 膜的羊膜和其余部分. </li>
	<li> 在胚胎的肱水平处切开横 SNT 段, 将尾延伸至胸廓区域以隔离 SNT ( 图 2 ). </li>
	<li> 移除腹周围 SNT ( (如 侧板胚层、外胚层、脊索和主动脉) 的组织, 使用镊子 ( 图 2 )。
	<li> 将每个解剖过的鸡 SNT 段浸泡在3.5 厘米的培养皿中, 其中含有5毫升的温热 (37 和 #176; C) 胰蛋白酶 (胰蛋白酶/EDTA 溶液 1:250). </li>
	<li> 在显微镜下检测到 SNT 周围性组织松动时, 停止胰蛋白酶治疗. 
	注意: trypsinization 时间必须严格控制, 以避免神经组织的 over-digestion 和分散, 并保持 SNT 的结构完整性. </li>
	<li> 胰蛋白酶处理后, 手动取出剩余的间质和外胚层组织, 准备干净的 SNT 切片. </li>
	<li> 将 SNT 部分转至1.5 毫升管, 并将其在液氮中冻结。一旦足够的 SNT 部分积累 (池 SNT 部分从〜30鸡胚为一个芯片反应), 存储在-80 和 #176; C. 
	注意: 如果有足够的胚胎组织 ( 即, 5 x 10 5 -5 x 10 6 细胞) 可以从一个或几个鸡胚中解剖, 那么冷冻是不必要的, 它可以直接进入下一步。请参阅 表 1 中的疑难解答指南. 
	注意: 小心!液氮具有极低的温度, 穿戴适当的保护. </li>
</ol> 2。交联蛋白到 DNA: 1 天 注意: 除非另有说明, 否则请执行所有的冰上步骤. 
<ol> <li> 添加500和 #181; DMEM 与 10% FBS 和 10 #181; l 1 M Na 丁酸酯直接到冷冻组织, 或者, 或者, 立即到刚解剖的组织。用1毫升吸管轻轻 re-suspending 质细胞. 
	注: 添加 Na 丁酸酯是可选的, 但建议如果调查组蛋白乙酰化。而不是 DMEM-10%fbs, 样品可以悬浮在 1x PBS 与 0.1% BSA。添加 FBS 或 BSA 是可选的, 但它有助于在随后的离心步骤中对样品进行制粒. </li>
	<li> 添加13.5 和 #181; L 37% 甲醛 (1% 甲醛决赛), 并把它放在一个转子15分钟的室温. 
	注意: 小心!甲醛是有毒的. </li>
	<li> 添加25和 #181; L 甘氨酸2.5 米的管, 并把它放在一个旋转10分钟的室温, 以淬火甲醛. </li>
	<li> 旋转管在 850 x g 为5分钟在4和 #176; C 在台式离心机。丢弃上清. </li>
	<li> 一次用500和 #181 清洗颗粒; 冷新鲜准备的洗涤缓冲器 (参见 补充材料 为食谱), 离心机在 850 x g 和4和 #176; C 为5分钟, 并丢弃上清. </li>
</ol> 3。裂解和超声 <ol> <li> 准备完整的裂解缓冲区 (LB) (参见 辅助材料 食谱), 然后冰镇。1毫升, 使用950和 #181; l 的 LB, 40 和 #181; l 的蛋白酶抑制剂浓缩物 (25x), 和10和 #181; l 100% PMSF. </li>
	<li> 重300和 #181 中的颗粒; L 由移完成 LB, 并将其放在一个摇摆平台上10分钟, 在4和 #176; C. </li>
	<li> 使用以下设置几种示例: Temp = 4 和 #176; C, 总时间 = 7 分钟, #8220; #8221; 间隔 = 三十年代, #8220; 关闭和 #8221; 间隔 = 三十年代, 振幅 = 25% 
	注意: 超声条件需要对每个组织进行优化, 并根据所使用的 sonicator 而有所不同。建议使用最低的超声设置, 导致 DNA 的剪切范围从200到 600 bp 的大小。剪切的差异很大, 取决于组织类型, 数量超声体积、交联条件和设备。请参阅 表 1 中的疑难解答指南. </li>
	<li> 将声样本在 1.6万 x g 上旋转10分钟, 在4和 #176; C 以颗粒状的细胞碎片. </li>
	<li> 将裂解液 (上清) 转换为新鲜的1.5 毫升管. 
	注: 如果开始材料被认为足够为二个芯片实验, 则300和 #181; 完全 LB 的 L 可以被增加到声染色质 ( 即, 最后的容量: 600 和 #181; l). </li>
	<li> 添加30和 #181; 10% 辛基乙烯每300和 #181 的 l. 声裂解产物 (最终浓度: 1%), 并由移混合. 
	注意: 小心!辛基乙烯有剧毒 (口腔、真皮、吸入). </li>
</ol> 4。抗体孵育 <ol> <li> 分330和 #181; l 声裂解为两个 1.5 mL 管: 300 和 #181; l 为芯片和30和 #181; l 作为输入控制 (起始材料的 10%)。存储 #34; 30、#181; 输入控制样本和 #34; 在-20 和 #176; C. 
	注意: 如果两个芯片反应是从同一个样品中进行的, 然后, 660 和 #181; l 声裂解物可分为三1.5 毫升管 (300 和 #181; l 为 ChIP#1, 300 和 #181; l 为 ChIP#2, 和60和 #181; l 作为输入控制 (每个芯片的起始材料的 20%)).</li>
	<li> 添加 3-5 和 #181; 300 和 #181 的抗体 g; 声染色质分和倒置混合. 
	注: 在本研究中, 每一个芯片反应都是在 K4 (H3K4me3), 组蛋白 H3 三甲基化的抗体特异性, 在 K4 (H3K4me2), 蛋白 H3 三甲基化, K27 (H3K27me3) 和组蛋白 H3 三乙酰化在 K27 (H3K27me3)。该协议的成功与否完全取决于所使用的抗体的质量。抗体应该是特定的和有效的沉淀特定的蛋白质。确定可用于 immunoprecipitate 交联蛋白-DNA 复合物的抗体量是非常重要的。此外, 抗体的特异性可以检查的免疫印迹, 以确保它检测到正确的蛋白质。不建议在芯片上检测多个非特异波段的抗体. </li>
	<li> 将导管放置在4和 #176 的摇摆平台上; C 过夜 (12-16 小时) 将抗体与染色质结合. 
	注意: 请参阅 表 1 中的疑难解答指南. </li>
</ol> 5。磁珠的制备: 2 天 <ol> <li> 分 50-100 和 #181; 蛋白质 G/磁珠浆料 L 为每个芯片反应成1.5 毫升离心管冰. </li>
	<li> 用冷块解决方案洗涤磁珠三次 (参见 辅助材料 为食谱) (4 和 #176; C)。加入500和 #181; L 冷块溶液, 并通过倒置或翻转混合。把管子放在磁性的支架上, 等待珠子在管子的侧面固定下来。把清澈的液体倒掉, 然后重复。最后一次洗涤后, 用凝胶加载的尖端去除所有液体. </li>
</ol> 6。染色质的沉淀 <ol> <li> 添加300和 #181; L 抗体结合染色质到洗涤的珠子和倒置混合. </li>
	<li> 在4和 #176 的管转子上垂直旋转; C 为至少4小时将抗体绑定到珠子. </li>
</ol> 7。清洗、洗和反转通道 <ol> <li> 冷帕洗涤缓冲器 (参见 补充材料 为食谱), 设置水浴到65和 #176; c, 冷离心机到4和 #176;
	<li> 在1毫升的冷帕洗涤缓冲液中洗涤染色质结合的珠子四次, 并保持在冰上。为此, 添加1毫升的冷帕洗涤缓冲和混合通过倒置或轻拂。把管子放到磁性的支架上, 等待珠子在管子的一侧固定下来。把清澈的液体倒掉, 然后重复. 
	注: 使用帕洗涤缓冲液的洗涤次数可能需要根据样品种类、样品丰度和抗体进行优化。冲洗次数的增加将减少背景, 但也会减少恢复的 DNA 总量, 这可能危及下游分析的 qPCR 或芯片 seq. </li>
	<li> 添加1毫升的 TE/50 毫米氯化钠到管, 并转移到一个新鲜的1.5 毫升离心管 TE/50 毫米氯化钠的染色质结合的珠子, 之前, 使用磁性持有人, 如上所述, 去除液体. </li>
	<li> 旋转 900 x g 的珠子为3分钟, 在4和 #176; C, 将管放回磁性支架, 并将所有剩余的 TE 与凝胶加载尖端一起移除. </li>
	<li> 在室温和重中添加210和 #181; 洗脱缓冲器的 L (参见 辅助材料 ). </li>
	<li> 洗在65和 #176 时为15分钟; 在摇晃时为热块. </li>
	<li> 在室温下旋转 1.6万 x g 的珠子1分钟, 并将管放入磁性支架中. </li>
	<li> 将上清液 (〜200和 #181; L) 转到一个新鲜的离心管, 一旦珠子已经解决. </li>
	<li> 将输入控制样本 (30 和 #181; l) 从-20 和 #176 解冻; C 到室温 (在步骤4.1 中冻结), 添加3卷洗脱缓冲液 (90 和 #181; l), 然后简单地涡流. </li>
	<li> 在65和 #176 上孵育芯片并控制样品; C 过夜 (12-16 小时) 以反转通道. </li>
</ol> 8。消化细胞蛋白和 RNA: 3 天 <ol> <li> 8.1At 室温, 增加1体积的 te 缓冲器每管 (稀释 SDS) 和倒置混合: 添加120和 #181; l 的 te 到120和 #181; l 的控制样品和200和 #181; l 的 te 到200和 #181; l 的芯片样本. </li>
	<li> 添加核糖核酸到0.2 毫克/毫升最终浓度和倒置混合: 添加2.4 和 #181; 20 毫克/毫升核糖核酸 a 到240和 #181; 控制样品 l 和4和 #181; 20 毫克/毫升核糖核酸 a 到400和 #181; l 的芯片样本. </li>
	<li> 在37和 #176 的试管中孵育; C 在水浴2小时, 以消化 RNA. </li>
	<li> 添加蛋白酶 K 到最终浓度为0.2 毫克/毫升和倒置混合: 添加2.4 和 #181; 20 毫克/毫升蛋白酶 k 到240和 #181; 控制样品 l 和4和 #181; 20 毫克/毫升蛋白酶 k 到400和 #181; 芯片样本的 l. </li>
	<li> 孵育在55和 #176; C 在水浴为 2 h 消化蛋白质和净化脱氧核糖核酸. </li>
</ol> 9。芯片 qPCR 注意: 执行 DNA 提取和纯化, 如 辅助材料 中所述。
 注意: 验证 "补充材料" 中所述的超声效率, 以确认获得200至 500 bp DNA 片段 ( 图 3 )。
 注意: 关键步骤是确定芯片是否实际工作。如果有已知的蛋白质的基因组结合点的兴趣, 引物可以设计定量 PCR (qPCR), 以确定是否已知的网站是具体丰富的芯片 DNA, 与负控制区域的预期不受约束甘地达萨日期蛋白质。作为一个例子, 这项工作显示了芯片 qPCR 的结果获得了 H3K4me3 (主动启动子组蛋白标记) 和 H3K27me3 (不活泼启动子组蛋白标记) 在 SNT 部分分离 HH14 小鸡胚胎 ( 图4A).
<ol> <li> 在同一管中混合每个引物对 (正向和反转), 并准备20和 #181 的库存浓度; M 每个使用 dH 2 O ( 表 2 )。
	注: 每个底漆对的效率应在芯片 qPCR 分析前进行评估. </li>
	<li> 使用步骤8.5 中获得的芯片和20% 输入控制 DNA 进行 qPCR 优化. 
	注: 输入的 DNA 通常是稀释的 20x (1% 的起始材料用于芯片反应) 进行 qPCR 分析. </li>
	<li> 每个10和 #181; pcr, 将下列成分混合在 pcr 管中: 用于 dna mastermix, 添加2和 #181; dH2O、2.5 和 #181; l SYBR 绿色混合, 1 和 #181; l 的 dna (从芯片或1% 输入); mastermix 与底漆, 增加2.375 和 #181; dH2O、2.5、#181; SYBR 绿色混合, 0.125 和 #181; 前向和反向底漆混合的 l. </li>
	<li> 将样品混合涡流 2 s, 并在 900 x g 处短暂离心, 1 分钟. </li>
	<li> 执行 real-time PCR (此处使用的引物和循环条件的示例, 请分别在 表 2 和 表 3 中找到). 
	注: 芯片 qPCR 数据分析: 芯片信号被计算为输入的百分比。简要地, 一旦获得的每 qPCR 反应的 ct 值, 一个稀释因子100或6.644 周期 (log2 100) 被应用于获得的 ct 值的输入 DNA 反应。然后, 对于给定的感兴趣区域 ( 即, 底漆对), 芯片信号的计算方法如下: 调整后的输入 = ct (输入)-6.644 芯片信号 = 100 x 电源 (2; 调整后输入的平均值-ct 值芯片样本) </li> </ol>
 10. 芯片-seq 库的准备;结束修复: 4 天 <ol> <li> 在60和 #181 中稀释100的芯片 DNA; 洗脱缓冲区的 L (请参阅 材料 表 ). </li>
	<li> 添加40和 #956; 结束修复混合和吸管轻轻10次. #160; </li>
	<li> 在热循环上孵育30和 #176; C 为30分钟. </li>
	<li> 涡流磁珠, 并添加160和 #956; L 到含有末端修复组合的管子。移10次彻底混合。室温孵育15分钟. </li>
	<li> 将管放入磁性支架, 等待珠子在管子的一侧固定.. </li>
	<li> 倒掉清澈的液体. </li>
	<li> 在将导管保持在磁性支架上的同时, 添加200和 #956; 我刚准备好80% 乙醇. </li>
	<li> 在室温下孵育三十年代并丢弃上清液。重复一次.. </li>
	<li> 在室温下保持15分钟的磁管。一旦颗粒干燥, 从磁性支架上取出管子. </li>
	<li> 重20和 #956 中的颗粒; 悬浮缓冲的 L。轻轻吸管10次混合彻底. </li>
	<li> 室温孵育2分钟. </li>
	<li> 将管放入磁性支架, 等待珠子在管子的一侧固定。转移17.5 和 #956; 升上清到一个新的管. </li>
</ol> 11。芯片-seq 库的准备;腺苷酸3和 #39; 结束 <ol> <li> 添加12.5 和 #956; L 尾砂混合到新的管和 #160; 通过轻轻移上下10次, 混合彻底。将管置于热循环上, 并按照以下程序进行孵化:37、#176; c 为30分钟、70和 #176; c 为5分钟, 并在4和 #176 中举行; c </li> </ol> 12。芯片-seq 库的准备;结扎适配器 <ol> <li> 添加2.5 和 #956; 悬浮缓冲区、2.5 和 #956、结扎混合的 l 和2.5 和 #956; 适当的 RNA 适配器索引 l。通过轻轻移上下10次, 混合彻底. </li>
	<li> 在30和 #176 的热循环上孵育10分钟;. </li>
	<li> 添加5和 #956; 停止结扎缓冲液, 并 #160 彻底混合; 移10次. </li>
	<li> 涡流磁珠, 并添加42.5 和 #956; L 到样本。移和 #160 彻底混合; 10 次。室温孵育15分钟. </li>
	<li> 将管放入磁性支架, 等待珠子在管子的一侧固定, 然后倒出清澈的液体 </li>
	<li> 在将导管保持在磁性支架上的同时, 添加200和 #956; 我刚准备好80% 乙醇. </li>
	<li> 在室温下孵育三十年代并丢弃上清液。重复一次. </li>
	<li> 将导管保持在磁性支架上, 让样品风干15分钟. </li>
	<li> 从磁性支架上取出管, 并在52.5 和 #956 中重颗粒; 悬浮缓冲器的 L。移10次彻底混合。在室温下孵育2分钟, 把管子放到磁性支架上, 等待珠子在管子的侧面沉淀。转移50和 #956; 我的清上清到一个新的管. </li>
	<li> 涡流磁珠, 并添加50和 #956; L 到样本。移和 #160 彻底混合; 10 次。室温孵育15分钟. </li>
	<li> 将管放在磁性支架上, 等待珠子在管子的一侧固定, 然后倒出清澈的液体. </li>
	<li> 在将导管保持在磁性支架上的同时, 添加200和 #956; 我刚准备好80% 乙醇. </li>
	<li> 在室温下孵育三十年代并丢弃上清液。重复一次. </li>
	<li> 将管保持在磁性支架上, 让样品风干15分钟. </li>
	<li> 从磁性支架上取出管, 并在27.5 和 #956 中重颗粒; 悬浮缓冲器的 L。移10次彻底混合. </li>
	<li> 室温孵育2分钟, 将管子放入磁性支架, 等待珠子在管子的一侧固定。转移25和 #956; 我的清上清到一个新的管. </li>
</ol> 13。芯片-seq 库的准备;DNA 片段的放大 <ol> <li> 放置12.5 和 #181; 在0.2 毫升的 PCR 管中的模板 L。添加2.5 和 #956; pcr 引物鸡尾酒, 10 和 #956; 增强 pcr 混合的 l. 和 #160; 通过移上下10次彻底混合。将管道置于热循环上, 并使用 表 4 中描述的条件. </li>
	<li> 涡流磁珠, 并添加25和 #956; L 到样本。移和 #160 彻底混合; 10 次。室温孵育15分钟. </li>
	<li> 将管放入磁性支架, 等待珠子在管子的一侧固定, 然后倒入清澈的液体. </li>
	<li> 在将导管保持在磁性支架上的同时, 添加200和 #956; 我刚准备好80% 乙醇。在室温下孵育三十年代并丢弃上清液。重复一次. </li>
	<li> 将导管保持在磁性支架上, 并让样品风干15分钟. 从磁性支架上取出导管, 并在32.5 和 #956 中重颗粒; 悬浮缓冲器的 L。移10次彻底混合. </li>
	<li> 孵化在室温下为2分钟的 PCR 管/板。将 PCR 管/板放在室温下的磁性支架上, 等待珠子在试管的一侧沉淀, 然后转移30和 #956; 升上清到一个新的管. </li>
</ol> 14。芯片-seq 库的准备;库验证 注意: 使用 DNA 和 RNA 质量控制系统对库进行验证。梯子的准备: 分1和 #181; l 基因组 DNA 阶梯进入第一管/井, 并添加3和 #181; 示例缓冲区的 l. 
<ol> <li> 示例的准备: 混合1和 #181; 库的 l 示例 (10-100 ng/和 #181; l) 与3和 #181; 在试管中取样缓冲器的 l。旋转下来, 然后在最大速度涡旋 5 s. 向下旋转以将样品放置在管子的底部. </li>
	<li> 将示例加载到质量控制系统中. </li>
	<li> 运行完成后, 通过设置最大值的对比度来分析结果, 以确保示例中不存在入门聚 ( 图 5 ); 底漆聚应该对应于一个波段约 135 bp。如果连弱频带都存在, 则通过添加洗脱缓冲区 (参见 材料表 ) 进行第二次清理, 其容量可达50和 #181; l 体积和添加47.5 和 #181; 混合磁珠的 l。如果样品的浓度太低 (和 #60; 2 nM), 继续运行 PCR (步骤 13.1), 使用18周期而不是 15, 与12.5 和 #181; L 的样本量从步骤12.16 中离开. </li>
	<li> 使用商用工具包 qPCR 单独量化库. 
	注意: 可以使用 single-read 1 x 50 nt 协议的排序器对库池进行排序, 其目标为30米读/样. </li>
</ol>

<ol>
	<li>Jenuwein, T., Allis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+the+histone+code.">Translating the histone code.</a> Science. 293 (5532), 1074-1080 (2001).</li><li>Kouzarides, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+modifications+and+their+function.">Chromatin modifications and their function.</a> Cell. 128 (4), 693-705 (2007).</li><li>Wang, Z., Schones, D. E., Zhao, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+human+epigenomes.">Characterization of human epigenomes.</a> Curr Opin. Genet Dev. 19 (2), 127-134 (2009).</li><li>ENCODE Project Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+encyclopedia+of+DNA+elements+in+the+human+genome.">An integrated encyclopedia of DNA elements in the human genome.</a> Nature. 489 (7414), 57-74 (2012).</li><li>Roadmap Epigenomics Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+analysis+of+111+reference+human+epigenomes.">Integrative analysis of 111 reference human epigenomes.</a> Nature. 518 (7539), 317-330 (2015).</li><li>Heintzman, N. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+modifications+at+human+enhancers+reflect+global+cell-type-specific+gene+expression.">Histone modifications at human enhancers reflect global cell-type-specific gene expression.</a> Nature. 459 (7243), 108-112 (2009).</li><li>Rada-Iglesias, A., Bajpai, R., Swigut, T., Brugmann, S. A., Flynn, R. A., Wysocka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+chromatin+signature+uncovers+early+developmental+enhancers+in+humans.">A unique chromatin signature uncovers early developmental enhancers in humans.</a> Nature. 470 (7333), 279-283 (2011).</li><li>Creyghton, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+H3K27ac+separates+active+from+poised+enhancers+and+predicts+developmental+state.">Histone H3K27ac separates active from poised enhancers and predicts developmental state.</a> Proc Nat Acad Sci USA. 107 (50), 21931-21936 (2010).</li><li>Benayoun, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H3K4me3+breadth+is+linked+to+cell+identity+and+transcriptional+consistency.">H3K4me3 breadth is linked to cell identity and transcriptional consistency.</a> Cell. 158 (3), 673-688 (2014).</li><li>Rehimi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenomics-Based+Identification+of+Major+Cell+Identity+Regulators+within+Heterogeneous+Cell+Populations.">Epigenomics-Based Identification of Major Cell Identity Regulators within Heterogeneous Cell Populations.</a> Cell Rep. 17 (11), 3062-3076 (2016).</li><li>Whyte, W. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Master+transcription+factors+and+mediator+establish+super-enhancers+at+key+cell+identity+genes.">Master transcription factors and mediator establish super-enhancers at key cell identity genes.</a> Cell. 153 (2), 307-319 (2013).</li><li>Valouev, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+transcription+factor+binding+sites+based+on+ChIP-Seq+data.">Genome-wide analysis of transcription factor binding sites based on ChIP-Seq data.</a> Nat Methods. 5 (9), 829-834 (2008).</li><li>Dahl, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broad+histone+H3K4me3+domains+in+mouse+oocytes+modulate+maternal-to-zygotic+transition.">Broad histone H3K4me3 domains in mouse oocytes modulate maternal-to-zygotic transition.</a> Nature. 537 (7621), 548-552 (2016).</li><li>Rotem, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+ChIP-seq+reveals+cell+subpopulations+defined+by+chromatin+state.">Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state.</a> Nat Biotech. 33 (11), 1165-1172 (2015).</li><li>Schmidl, C., Rendeiro, A. F., Sheffield, N. C., Bock, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIPmentation:+fast,+robust,+low-input+ChIP-seq+for+histones+and+transcription+factors.">ChIPmentation: fast, robust, low-input ChIP-seq for histones and transcription factors.</a> Nat Methods. 12 (10), 963-965 (2015).</li><li>Zhang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allelic+reprogramming+of+the+histone+modification+H3K4me3+in+early+mammalian+development.">Allelic reprogramming of the histone modification H3K4me3 in early mammalian development.</a> Nature. 537 (7621), 553-557 (2016).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> Dev Dynam. 195 (4), 231-272 (1951).</li><li>Ho, J. W. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+metazoan+chromatin+organization.">Comparative analysis of metazoan chromatin organization.</a> Nature. 512 (7515), 449-452 (2014).</li><li>Calo, E., Wysocka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+enhancer+chromatin:+what,+how,+and+why?.">Modification of enhancer chromatin: what, how, and why?.</a> Mol Cell. 49 (5), 825-837 (2013).</li><li>Spitz, F., Furlong, E. E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factors:+from+enhancer+binding+to+developmental+control.">Transcription factors: from enhancer binding to developmental control.</a> Nat Rev Genetics. 13 (9), 613-626 (2012).</li></ol>

作者没有任何相互竞争的金融利益有待披露。

表组蛋白修饰的芯片序列可以用于改善不同细胞背景下的脊椎动物的功能性注释4,5,18。除其他外, 这些表配置文件可用于确定增强剂元素, 以定义促进剂的调节状态 (即,活动、引物或准备), 以及定义不同生物或病理环境 (例如,广泛的 H3K4me3 启动子域和 super-enhancers)6,7,9,10,11,19。然而, 由于芯片分析的固有的局限性, 通常需要数百万个细胞, 大多数组蛋白修饰的配置文件都是在体外细胞系和细胞类型, 可以被排序在体内大量4. 最近, 一些芯片序列协议被描述为与极低的细胞数兼容, 从而极大地扩大了细胞类型和组织的范围, 其中表配置文件原则上可以生成13 ,14,15,16。这些新的芯片 seq 协议都依赖于特定的ad hoc库准备方法, 它们至少在某些情况下需要非标准设备和/或试剂14、15、16。
在这里, 我们描述一个芯片协议的工作与低到中间的细胞数 (5 x 104 -5 x 105细胞) 获得在体内从不同的胚胎组织10。为了提高芯片检测的灵敏度, 我们已经消除了芯片中通常使用的几个步骤, 如顺序电池和核裂解, 这会导致有价值的物质逐渐流失。因此, 在交联后, 样品用单一的裂解缓冲剂处理, 随后声以尽量减少样品损失。重要的是, 我们的芯片协议是兼容的 qPCR 分析, 从而使表分析选定的兴趣点。此外, 我们的芯片协议可以与标准的芯片 seq 库的准备方法相结合, 从而对大多数能够获得下一代测序技术的实验室都有潜在的帮助。
芯片和芯片序列协议不仅可以用来研究组蛋白修饰的结合型, 还能揭示转录因子和 co-activators 等其他重要转录调控器的结合位点 (例如,p300 和 CBP)20。通常, 芯片 (和芯片序列) 的转录因子和 co-activators, 不像组蛋白, 需要相当多的起始材料比芯片组组标记。因此, 除非启动单元格的数量大大增加 (例如, 5 x 105 -5 x 106单元格) 和/或高度特定的, 否则所描述的协议可能对大多数转录因子或 co-activators 都不起作用有效的抗体是可用的。然而, 随着进一步优化和不断改进的图书馆准备方法, 我们预计, 芯片序列从有限的蜂窝材料将成为可行的大多数调节蛋白。
虽然我们的芯片和芯片序列协议已经得到了广泛的验证, 为各种组蛋白修饰和胚胎组织, 我们想强调一些关键步骤, 我们认为是必不可少的, 以产生高质量的表配置文件。首先, 在不损害染色质完整性的条件下, 胚胎标本需要新鲜的隔离。这些包括在寒冷的条件下进行解剖, 或在有足够的材料积累之前在冰冻的样品中进行。另一个关键步骤是超声, 需要精确地优化以执行高质量的芯片。根据组织类型、组织数量或所使用的 sonicator, 最佳超声条件经常变化。最后但并非最不重要的是, 成功的芯片最终依赖于特定和芯片相容的抗体对蛋白质的兴趣。

组蛋白修饰修饰直接涉及各种染色质相关的过程, 包括转录, 复制和 DNA 修复1,2,3。此外, 不同的组蛋白修饰显示阳性 (例如, H3K4me3 和 H3K27ac) 或阴性 (例如, H3K9me3 和 H3K27me3) 与基因表达的相关性, 可以被广泛定义为激活或压制组蛋白标记,分别为2,3。因此, 全球组蛋白修饰地图, 也称为表地图, 已成为功能强大的和通用的工具, 对脊椎动物的基因,4,5。例如, 可以根据特定染色质特征 (例如,活性增强剂: H3K4me1 和 H3K27ac) 来识别远端调节序列 (如增强剂), 使其与近端启动子区域 (例如活动发起人: H3K4me3)6,7,8。另一方面, 具有主要细胞身份调节功能的基因通常在具有 H3K4me3 或 H3K27me3 标记的广泛的染色质域中发现, 这取决于基础基因的转录活性或不活泼状态, 分别为9 ,10。同样, 主要细胞标识基因的表达似乎经常由多重和空间聚类增强剂 (即, super-enhancers) 控制, 可被确定为广泛的 H3K27ac-marked 域11。
目前, 组蛋白修饰图是使用芯片-seq 技术生成的, 这与以前的方法 (芯片耦合到微阵列) 相比, 提供了更高的分辨率、更少的工件、更少的噪音、更大的覆盖率和更低的成本12。然而, 使用芯片 seq 技术生成的表映射有其固有的局限性, 主要与在感兴趣的样本中成功执行芯片的能力有关。传统的芯片协议通常需要数以百万计的细胞, 这就限制了这种方法对体外细胞系或细胞的适用性. 在过去的几年里, 一些修改过的芯片协议与低的细胞数兼容被描述了13,14,15,16。但是, 这些协议专门设计为与下一代序列 (即,芯片 seq) 结合使用, 并且它们通常采用ad hoc库准备方法13,14,15,16。
在这里, 我们描述了一个芯片协议, 可用于调查组蛋白修饰配置文件使用低到中间的细胞数 (5 x 104 -5 x 105细胞) 在任一选定的所在地 (即qPCR) 或全局 (即芯片-seq) (图 1)。当耦合到芯片 seq 技术时, 我们的芯片协议可以与标准库的准备方法一起使用, 从而使许多实验室10得到广泛的访问。该协议已被用来调查几个组蛋白标记(例如, H3K4me3, H3K27me3 和 H3K27ac) 在不同的鸡胚组织 (例如,脊髓神经管 (SNT), 额日珥, 和叶)。然而, 我们预计, 它应广泛适用于其他生物体, 其中生物和/或临床相关样品只能获得低量。

<table>
	<tbody>
		<tr>
			<td>Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BSA powder</td>
			<td>Carl Roth</td>
			<td>3737.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Saline buffer (PBS)</td>
			<td>Sigma Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCL pH8.0</td>
			<td>Sigma Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Carl Roth</td>
			<td>3957.2</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Carl Roth</td>
			<td>8043.2</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Carl Roth</td>
			<td>3054.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Na-Deoxycholate</td>
			<td>Sigma Aldrich</td>
			<td>D6750-24</td>
			<td></td>
		</tr>
		<tr>
			<td>N-lauroylsarcosine</td>
			<td>Sigma Aldrich</td>
			<td>61743-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Applichem</td>
			<td>A3724,0250</td>
			<td></td>
		</tr>
		<tr>
			<td>LiCl</td>
			<td>Carl Roth</td>
			<td>3739.2</td>
			<td></td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Sigma Aldrich</td>
			<td>I3021-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Carl Roth</td>
			<td>1833</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein G/magnetic beads</td>
			<td>Invitrogen</td>
			<td>1004D</td>
			<td></td>
		</tr>
		<tr>
			<td>37% Formaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>252549-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RNase</td>
			<td>Peqlab</td>
			<td>12-RA-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Sigma Aldrich</td>
			<td>46.35 E</td>
			<td></td>
		</tr>
		<tr>
			<td>Na-butyrate</td>
			<td>Sigma Aldrich</td>
			<td>SLB2659V</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase inhibitor</td>
			<td>Roche</td>
			<td>5892791001</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBRgreen Mix</td>
			<td>biozym</td>
			<td>617004</td>
			<td></td>
		</tr>
		<tr>
			<td>dH2O</td>
			<td>Sigma Aldrich</td>
			<td>W4502</td>
			<td></td>
		</tr>
		<tr>
			<td>1Kb ladder</td>
			<td>Thermofisher</td>
			<td>SM1333</td>
			<td></td>
		</tr>
		<tr>
			<td>Orange G</td>
			<td>Sigma Alrich</td>
			<td>03756-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Invitrogen</td>
			<td>16500-500</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA (1X)</td>
			<td>Gibco</td>
			<td>25200-072</td>
			<td></td>
		</tr>
		<tr>
			<td>Octylphenol Ethoxylate (Triton X 100)</td>
			<td>Roth</td>
			<td>3051.4</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (Dulbecco&#180;s Modified Eagle Medium)</td>
			<td>Gibco</td>
			<td>31331-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel loading tips Multiflex</td>
			<td>A.Hartenstein</td>
			<td>GS21</td>
			<td></td>
		</tr>
		<tr>
			<td>qPCR Plates</td>
			<td>Sarstedt</td>
			<td>721,985,202</td>
			<td></td>
		</tr>
		<tr>
			<td>384 well</td>
			<td>Sarstedt</td>
			<td>721,985,202</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL tubes</td>
			<td>Sarstedt</td>
			<td>72,706</td>
			<td></td>
		</tr>
		<tr>
			<td>100-1000&#181;l Filter tips</td>
			<td>Sarstedt</td>
			<td>70,762,211</td>
			<td></td>
		</tr>
		<tr>
			<td>2-20&#181;l Filter tips</td>
			<td>Sarstedt</td>
			<td>70,760,213</td>
			<td></td>
		</tr>
		<tr>
			<td>2-200&#181;l Filter tips</td>
			<td>Sarstedt</td>
			<td>70,760,211</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5-10&#181;l Filter tips</td>
			<td>Sarstedt</td>
			<td>701,116,210</td>
			<td></td>
		</tr>
		<tr>
			<td>H3K27me3 antibody</td>
			<td>Active Motif</td>
			<td>39155</td>
			<td></td>
		</tr>
		<tr>
			<td>H3K27ac antibody</td>
			<td>Active Motif</td>
			<td>39133</td>
			<td></td>
		</tr>
		<tr>
			<td>H3K4me3 antibody</td>
			<td>Active Motif</td>
			<td>39159</td>
			<td></td>
		</tr>
		<tr>
			<td>H3K4me2 antibody</td>
			<td>Active Motif</td>
			<td>39141</td>
			<td></td>
		</tr>
		<tr>
			<td>End Repair Mix</td>
			<td>Illumina</td>
			<td>FC-121-4001</td>
			<td></td>
		</tr>
		<tr>
			<td>Paramagnetic beads</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
			<td></td>
		</tr>
		<tr>
			<td>Resuspension buffer</td>
			<td>Illumina</td>
			<td>FC-121-4001</td>
			<td></td>
		</tr>
		<tr>
			<td>A-Tailing Mix</td>
			<td>Illumina</td>
			<td>FC-121-4001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ligation Mix</td>
			<td>Illumina</td>
			<td>FC-121-4001</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA Adapter Indexes</td>
			<td>Illumina</td>
			<td>RS-122-2101</td>
			<td></td>
		</tr>
		<tr>
			<td>Stop Ligation Buffer</td>
			<td>Illumina</td>
			<td>FC-121-4001</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Primer Cocktail</td>
			<td>Illumina</td>
			<td>FC-121-4001</td>
			<td></td>
		</tr>
		<tr>
			<td>Enhanced PCR Mix</td>
			<td>Illumina</td>
			<td>FC-121-4001</td>
			<td></td>
		</tr>
		<tr>
			<td>Genomic DNA ladder</td>
			<td>Agilent</td>
			<td>5067-5582</td>
			<td></td>
		</tr>
		<tr>
			<td>Elution Buffer</td>
			<td>Agilent</td>
			<td>19086</td>
			<td></td>
		</tr>
		<tr>
			<td>Sample Buffer</td>
			<td>Agilent</td>
			<td>5067-5582</td>
			<td></td>
		</tr>
		<tr>
			<td>Library Quantification Kit</td>
			<td>Kapa Biosystems</td>
			<td>KK4835 &#8211; 07960204001</td>
			<td></td>
		</tr>
		<tr>
			<td>Fertile chicken white eggs</td>
			<td>LSL Rhein Main</td>
			<td>KN: 15968</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle (Neoject)</td>
			<td>A.Hartenstein</td>
			<td>10001</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe (Ecoject 10ml)</td>
			<td>Dispomed witt oHG</td>
			<td>21010</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Hermle</td>
			<td>Z 216 MK</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermoshaker</td>
			<td>ITABIS</td>
			<td>MKR13</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Active Motive</td>
			<td>EpiShear probe sonicator</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Diagenode</td>
			<td>Bioruptor Plus</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotator</td>
			<td>Stuart</td>
			<td>SB3</td>
			<td></td>
		</tr>
		<tr>
			<td>Variable volume (5&#8211;1,000 &#956;l) pipettes</td>
			<td>Eppendorf</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Timer</td>
			<td>Sigma</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic holder</td>
			<td>Thermo Fisher</td>
			<td>DynaMag-2 12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>Table spinner</td>
			<td>Heathrow Scientific</td>
			<td>Sprout</td>
			<td></td>
		</tr>
		<tr>
			<td>Mixer</td>
			<td>LMS</td>
			<td>VTX 3000L</td>
			<td></td>
		</tr>
		<tr>
			<td>Real-Time PCR Cycler</td>
			<td>Roche</td>
			<td>Light Cycler; Serial Nr.5662</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Cycler</td>
			<td>Applied Biosystems</td>
			<td>Gene Amp PCR System 9700</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA and RNA quality control system</td>
			<td>Agilent</td>
			<td>Agilent 4200 TapeStation System</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Dumont</td>
			<td>5-Inox-H</td>
			<td></td>
		</tr>
		<tr>
			<td>Perforated spoon</td>
			<td>World precision instruments</td>
			<td>501997</td>
			<td></td>
		</tr>
	</tbody>
</table>

chromatin immunoprecipitation (chip) protocol for low-abundance embryonic samples

染色质沉淀 (芯片) 是一种广泛使用的技术, 用于映射定位的 post-translationally 修饰组蛋白, 组织变异, 转录因子, 或染色质修饰酶在一个给定的位置或在整个基因范围的规模。芯片分析与下一代测序 (即,芯片序列) 的结合, 是一种在全球范围内发现基因调控网络的强有力的方法, 它可以改善染色体组的功能诠释, 特别是编码的调控序列。芯片协议通常需要大量的细胞材料, 从而排除了这种方法的适用性, 调查稀有细胞类型或小组织活检。为了使芯片检测与在早期脊椎动物胚胎发生过程中通常可以获得的生物材料的数量相一致, 我们在这里描述了一个简化的芯片协议, 其中需要完成的步骤数化验减少, 以尽量减少样本损失。该芯片协议已成功地用于调查不同的组蛋白修饰的各种胚胎鸡和成年小鼠组织使用低到中等细胞数 (5 x 104 -5 x 105单元格)。重要的是, 这个协议是兼容的芯片 seq 技术使用标准的库准备方法, 从而提供全球表地图在高度相关的胚胎组织。

为了说明我们的芯片协议的性能, 我们用 HH19 鸡胚、上颌日珥 HH22 鸡胚和 stage-HH3 鸡胚的 SNT 切片进行了芯片序列实验, 以探讨各种组蛋白修饰 (即H3K4me2、H3K27ac、H3K4me3 和 H3K27me3)。在获得芯片 dna 后, 用琼脂糖凝胶电泳对相应的输入 dna 进行了超声效率评估 (图 3)。然后, 芯片和输入的 dna 被用于芯片 qPCR 分析, 以测量富在座位上预期将被调查组蛋白的修改绑定或解除 (图 4A)。在图 4A中所示的示例中, HH19 SNT 节中的 H3K27me3 和 H3K4me3 富集级别分别在 qPCR 和 SOX17 的启动子区域中进行了芯片 SOX2 的评估, 两个基因在 SNT 中处于非活动状态和活动状态。如预期的那样, SOX2 启动子的强烈标记为 H3K4me3, 但不是由 H3K27me3, 而 SOX17 启动显示相反的模式 (图 4A)。通过对几个位点的评估, 可以估计出芯片的质量, 而不会丢失大量的 DNA 材料用于下游芯片分析。一旦确认了芯片的质量, 就准备好了芯片序列库并进行了排序。每个被调查的鸡胚组织都有代表性的基因座: (i) 在PAX7 (图 4B) 周围 HH19 鸡胚 SNT 的 H3K27me3 和 H3K4me3 的轮廓;(二) HH22 鸡胚上颌日珥 H3K4me2 和 H3K27ac 的剖面图在SNAI1 (4C) 周围;和 (iii) H3K27me3 在 HH3-stage 鸡胚周围 TBX3/TBX5 轨迹 (图 4D) 的剖面。这些芯片序列实验清楚地表明, 我们的芯片协议可以用来产生表剖面从有限数量的生物材料在广泛的胚胎组织。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56186/56186fig1.jpg"> 
图 1: 从鸡胚中分离的低丰度胚胎样品的芯片协议概述.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56186/56186fig1large.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/56186/56186fig2.jpg"> 
图 2: SNT 显微切割协议的简要概述.横 SNT 段在 HH19 鸡胚的肱水平处被切断。胰蛋白酶处理后, 性组织用镊子机械去除。不, 脊索;SNT, 脊髓神经管;所以, 节。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56186/56186fig2large.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/56186/56186fig3.jpg"> 
图 3: 最佳超声的示例.鸡 SNT HH19 样品是声为11个周期, 与三十年代和四十五年代关闭脉冲在 "高" 振幅使用 sonicator。通道被逆转, 纯化的 DNA 在1.5% 琼脂糖凝胶上得到了解决。在11周期后观察最佳片段大小, 范围从 200-500 bp.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56186/56186fig3large.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/56186/56186fig4.jpg"> 
图 4: 芯片分析的代表性例子 qPCR 或芯片-seq.(A) H3K27me3 (左) 和 H3K4me3 (右) 的水平在指定的区域是用芯片-qPCR 分析进行测量, 使用 SNT 分离的 HH19 鸡胚的部分。SOX2 启动子区域在 SNT 中是活跃的, 因此由 H3K4me3 而不是由 H3K27me3 标记;SOX17 启动子区域在 SNT 中处于非活动状态, 因此在 H3K27me3 中富集, 但不在 H3K4me3 中;Chr6 阴性代表一个基因区域在鸡染色体6并且担当阴性控制。误差线表示来自三技术复制的标准偏差。(B) 芯片序列 (H3K27me3 以洋红、蓝色 H3K4me3 和红色输入) SNTs 的 HH14 小鸡胚胎的轮廓显示在 PAX7 轨迹的周围。(C) 芯片序列 (H3K4me2 在橙色, H3K27ac 在绿色, 并输入红色) 从上颌日珥的 HH22 小鸡胚胎的轮廓显示在 SNAI1 的轨迹。(D) 芯片序列 (H3K27me3 在洋红色和输入红色) 剖面从 HH3 小鸡胚胎显示在 TBX3/TBX5 轨迹的周围。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56186/56186fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56186/56186fig5.jpg"> 
图 5: 使用自动化的 RNA 质量控制系统的芯片 seq 库验证示例.在 (A) 之前和 (B) 底漆二聚体清理后, 对相同的芯片序列库进行了自动化的 RNA 质量控制系统分析。底漆聚可以被看作是一个弱〜 135 bp 波段 (a)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56186/56186fig5large.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>步骤</td>
			<td>问题</td>
			<td>可能的原因</td>
			<td>解决</td>
		</tr>
		<tr>
			<td>12、34 5 及11</td>
			<td>根据芯片 qPCR 和/或芯片序列的低芯片信号到噪声比率</td>
			<td>组织质量受损。</td>
			<td>在寒冷的条件下进行显微解剖;否则染色质完整性可能会受到损害。</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>交联样品离心后样品损失。</td>
			<td>使用 DMEM 培养基与 10-20% 的血清或重复离心步骤, 增加时间到10分钟。</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>交叉链接的持续时间。</td>
			<td>优化交联时间。增加的交联时间可以促进对某些蛋白质的检测, 如 co-activators 和共同不直接与 DNA 结合。</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>PBS 中多次洗涤造成的样品损耗。</td>
			<td>减少洗涤次数;离心步骤可以重复在 1500 x g。</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>次优超声导致的 DNA 大小太长或太短。</td>
			<td>进行时间课程实验以优化超声条件。最佳片段大小, 从200到 500 bp</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>抗体不适合芯片。</td>
			<td>使用不同的抗体对内源蛋白或抗体对添加到外部表达的蛋白质 (例如标志, HA) 的标签。</td>
		</tr>
		<tr>
			<td>12</td>
			<td>qPCR 中的低或无信号, 甚至输入 DNA</td>
			<td>次优底漆</td>
			<td>检查底漆的效率和特异性;设计新的底漆。</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>DNA 数量不足</td>
			<td>没有足够的起始材料。增加每 qPCR 反应所使用的 DNA 数量。</td>
		</tr>
	</tbody>
</table>
-页 = "1" >表 1: 有关协议中涉及的各种步骤的疑难解答指南.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>引</td>
			<td>序列</td>
		</tr>
		<tr>
			<td>SOX2-F</td>
			<td>CCTTGCTGGGAGTACGACAT</td>
		</tr>
		<tr>
			<td>SOX2-R</td>
			<td>GCCCTGCAGTACAACTCCAT</td>
		</tr>
		<tr>
			<td>SOX17-F</td>
			<td>CCCTGAACTGTCATGTGTGG</td>
		</tr>
		<tr>
			<td>SOX17-R</td>
			<td>CAAACAGTTGCCTTTGAGCA</td>
		</tr>
		<tr>
			<td>Chr6-neg-F</td>
			<td>CCGTCAATCTCTGCTTGTGA</td>
		</tr>
		<tr>
			<td>Chr6-neg-R</td>
			<td>TGGAATCTGCTTGTCACTGC</td>
		</tr>
	</tbody>
</table>
表 2: 芯片 qPCR 的引物序列。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">Preincubation</td>
		</tr>
		<tr>
			<td>温度</td>
			<td colspan="2">时间</td>
		</tr>
		<tr>
			<td>95° c</td>
			<td colspan="2">5分钟</td>
		</tr>
		<tr>
			<td colspan="3">放大</td>
		</tr>
		<tr>
			<td>温度</td>
			<td colspan="2">时间</td>
		</tr>
		<tr>
			<td>95° c</td>
			<td>十年代</td>
			<td></td>
		</tr>
		<tr>
			<td>60° c</td>
			<td>十年代</td>
			<td>45</td>
		</tr>
		<tr>
			<td>72° c</td>
			<td>十年代</td>
			<td>周期</td>
		</tr>
		<tr>
			<td colspan="3">熔化曲线</td>
		</tr>
		<tr>
			<td>温度</td>
			<td colspan="2">时间</td>
		</tr>
		<tr>
			<td>95° c</td>
			<td colspan="2">5 s</td>
		</tr>
		<tr>
			<td>65° c</td>
			<td colspan="2">1分钟</td>
		</tr>
		<tr>
			<td colspan="3">冷却</td>
		</tr>
		<tr>
			<td>40° c</td>
			<td colspan="2">三十年代</td>
		</tr>
	</tbody>
</table>
表 3: 芯片 qPCR 的 PCR 条件。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">初始变性</td>
		</tr>
		<tr>
			<td>温度</td>
			<td colspan="2">时间</td>
		</tr>
		<tr>
			<td>95° c</td>
			<td colspan="2">3分钟</td>
		</tr>
		<tr>
			<td colspan="3">放大</td>
		</tr>
		<tr>
			<td>温度</td>
			<td colspan="2">时间</td>
		</tr>
		<tr>
			<td>98° c</td>
			<td>二十年代</td>
			<td></td>
		</tr>
		<tr>
			<td>60° c</td>
			<td>十五年代</td>
			<td>15</td>
		</tr>
		<tr>
			<td>72° c</td>
			<td>三十年代</td>
			<td>周期</td>
		</tr>
		<tr>
			<td colspan="3">最终扩展</td>
		</tr>
		<tr>
			<td>温度</td>
			<td colspan="2">时间</td>
		</tr>
		<tr>
			<td>72° c</td>
			<td colspan="2">5 s</td>
		</tr>
		<tr>
			<td colspan="3">冷却</td>
		</tr>
		<tr>
			<td>4° c</td>
			<td colspan="2">举行</td>
		</tr>
	</tbody>
</table>
表 4: 在芯片序列库制备过程中扩增 DNA 片段的 PCR 条件。

Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples at JoVE.com

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

在这里, 我们描述了染色质沉淀 (芯片) 和芯片序列库的准备协议, 以产生全球表剖面从丰鸡胚胎标本。

低丰度胚胎标本的染色质沉淀 (芯片) 协议

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

chromatin-immunoprecipitation-chip-protocol-for-low-abundance

Nanyang Technological University

Research

JoVE Journal

Genetics

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

15.6K 次观看.  University of Cologne. 在这里, 我们描述了染色质沉淀 (芯片) 和芯片序列库的准备协议, 以产生全球表剖面从丰鸡胚胎标本。

视频: Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous nature of most mammalian chromatin-modifying complexes. In order to overcome these hurdles, we have adapted a sequential salt extraction (SSE) assay for evaluating the relative binding affinities of chromatin-bound complexes. This easy and straightforward assay can be used by non-experts to evaluate the relative difference in binding affinity of two related complexes, the changes in affinity of a complex when a subunit is lost or an individual domain is inactivated, and the change in binding affinity after alterations to the chromatin landscape. By sequentially re-suspending bulk chromatin in increasing amounts of salt, we are able to profile the elution of a particular protein from chromatin. Using these profiles, we are able to determine how alterations in a chromatin-modifying complex or alterations to the chromatin environment affect binding interactions. Coupling SSE with other in vitro and in vivo assays, we can determine the roles of individual domains and proteins on the functionality of a complex in a variety of chromatin environments.

Sequential salt extraction of chromatin bound proteins is a useful tool for determining the binding properties of large protein complexes. This method can be employed to evaluate the role of individual subunits or domains in the overall affinity of a protein complex to bulk chromatin.

序贯盐萃取法分析染色质修饰配合物的体染色质结合特性

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous ...

科研

遗传学

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. Therefore, DNA-protein interactions regulate multiple physiological, pathophysiological, and biological functions, such as cell differentiation, cell proliferation, cell cycle control, chromosome stability, epigenetic gene regulation, and cell transformation. In eukaryotic cells, the DNA interacts with histone and nonhistone proteins and is condensed into chromatin. Several technical tools can be used to analyze DNA-protein interactions, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting. However, these techniques analyze the protein-DNA interaction in vitro, not within the cellular context. Chromatin immunoprecipitation (ChIP) is a technique that captures proteins at their specific DNA binding sites, thereby allowing for the identification of DNA-protein interactions within their chromatin context. It is done by fixation&#160;of the DNA-protein interaction, followed by&#160;immunoprecipitation of the protein of interest. Subsequently, the genomic site that the protein was bound to is characterized. Here, we describe and discuss ChIP and demonstrate its analytical value for the identification of the Transforming Growth Factor-&#946; (TGF-&#946;)-induced binding of the transcription factor SMAD2 to SMAD Binding Elements (SBE) within the promoter region of the tyrosine-protein kinase Kit (c-KIT) receptor ligand Stem Cell Factor (SCF).

DNA-protein interactions are essential for multiple biological processes. During the evaluation of cellular functions, the analysis of DNA-protein interactions is indispensable for understanding gene regulation. Chromatin immunoprecipitation (ChIP) is a powerful tool to analyze such interactions in vivo.

使用染色质免疫沉淀分析c-KIT配体启动子

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

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 ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

核密度分析的天然染色质沉淀测序文库的生成

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes that add or remove these marks in response to signals received by the cell. These PTMS are key contributors to the regulation of processes such as gene expression control and DNA repair. Chromatin immunoprecipitation (chIP) has been an instrumental approach for dissecting the abundance and localization of many histone PTMs throughout the genome in response to diverse perturbations to the cell. Here, a versatile method for performing chIP of post-translationally modified histones from the budding yeast Saccharomyces cerevisiae (S. cerevisiae) is described. This method relies on crosslinking of proteins and DNA using formaldehyde treatment of yeast cultures, generation of yeast lysates by bead beating, solubilization of chromatin fragments by micrococcal nuclease, and immunoprecipitation of histone-DNA complexes. DNA associated with the histone mark of interest is purified and subjected to quantitative PCR analysis to evaluate its enrichment at multiple loci throughout the genome. Representative experiments probing the localization of the histone marks H3K4me2 and H4K16ac in wildtype and mutant yeast are discussed to demonstrate data analysis and interpretation. This method is suitable for a variety of histone PTMs and can be performed with different mutant strains or in the presence of diverse environmental stresses, making it an excellent tool for investigating changes in chromatin dynamics under different conditions.

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Here, we describe a protocol for chromatin immunoprecipitation of modified histones from the budding yeast Saccharomyces cerevisiae. Immunoprecipitated DNA is subsequently used for quantitative PCR to interrogate the abundance and localization of histone post-translational modifications throughout the genome.

从酿酒酵母中组蛋白修饰的染色质沉淀 (芯片)

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

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 ...

Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of a wide range of transcription factors (TFs) and cofactors mediating transcriptional activation or repression via changes in chromatin structure. Chromatin immunoprecipitation (ChIP) is a useful molecular biology approach for mapping histone modifications and profiling transcription factors/cofactors binding to DNA, thus providing a snapshot of the dynamic nuclear changes occurring during different biological processes.
To study transcriptional regulation in adipose tissue, samples derived from in vitro cell cultures of immortalized or primary cell lines are often favored in ChIP assays because of the abundance of starting material and reduced biological variability. However, these models represent a limited snapshot of the actual chromatin state in living organisms. Thus, there is a critical need for optimized protocols to perform ChIP on adipose tissue samples derived from animal models.
Here we describe a protocol for efficient ChIP-seq of both histone modifications and non-histone proteins in brown adipose tissue (BAT) isolated from a mouse. The protocol is optimized for investigating genome-wide localization of proteins of interest and epigenetic markers in the BAT, which is a morphologically and physiologically distinct tissue amongst fat depots.

Here we describe a protocol for efficient chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) of brown adipose tissue (BAT) isolated from a mouse. This protocol is suitable for both mapping histone modifications and investigating genome-wide localization of non-histone proteins of interest in vivo.

小鼠棕色脂肪组织的染色质免疫沉淀

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of ...

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 ...

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 ...

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing (ChIP-seq)

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a technique that can be used to discover the regulatory targets of transcription factors, histone modifications, and other DNA-associated proteins. ChIP-seq data can also be used to find differential binding of transcription factors in different environmental conditions or cell types. Initially, ChIP was performed through hybridization on a microarray (ChIP-chip); however, ChIP-seq has become the preferred method through technological advancements, decreasing financial barriers to sequencing, and massive amounts of high-quality data output. Techniques of performing ChIP-seq with bacterial biofilms, a major source of persistent and chronic infections, are described in this protocol. ChIP-seq is performed on Salmonella enterica serovar Typhimurium biofilm and planktonic cells, targeting the master biofilm regulator, CsgD, to determine differential binding in the two cell types. Here, we demonstrate the appropriate amount of biofilm to harvest, normalizing to a planktonic control sample, homogenizing biofilm for cross-linker access, and performing routine ChIP-seq steps to obtain high quality sequencing results.

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing ChIP-seq

Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) is a method used to establish interactions between transcription factors and the genomic sequences they control. This protocol outlines techniques for performing ChIP-seq with bacterial biofilms, using Salmonella enterica serovar Typhimurium bacterial biofilm as an example.

使用染色质免疫沉淀和高通量测序（ChIP-seq）发现沙门氏菌生物膜中的CsgD监管目标

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a technique that can be used to discover the regulatory targets of transcription ...