Name: chromatin immunoprecipitation (chip) protocol for low-abundance embryonic samples
Uploaded: 2017-08-29T14:00:00.000+00:00
Duration: 12 min 47 s
Description: Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples at JoVE.com

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>

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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,<...

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>&lt;strong&gt;Reagent&lt;/strong&gt;</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 pH 8.0</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>dH&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich</td><td>W4502</td><td></td></tr><tr><td>1 Kb 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&amp;acute;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 - 1,000 &amp;micro;L Filter tips</td><td>Sarstedt</td><td>70,762,211</td><td></td></tr><tr><td>2 - 20 &amp;micro;L Filter tips</td><td>Sarstedt</td><td>70,760,213</td><td></td></tr><tr><td>2 - 200 &amp;micro;L Filter tips</td><td>Sarstedt</td><td>70,760,211</td><td></td></tr><tr><td>0.5 - 10 &amp;micro;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 &amp;ndash; 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 10 mL)</td><td>Dispomed witt oHG</td><td>21010</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</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 &amp;ndash;1,000 &amp;mu;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?...

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

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

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and organ formation during development. Determining the locations and levels of such chromatin features in vivo will provide valuable information about the spatio-temporal regulation of genomic elements, and will support aspirations to mimic embryonic tissue development in vitro. The most commonly used method for genome-wide and high-resolution profiling is chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq). This protocol outlines how yolk-rich embryos such as those of the frog Xenopus can be processed for ChIP-Seq experiments, and it offers simple command lines for post-sequencing analysis. Because of the high efficiency with which the protocol extracts nuclei from formaldehyde-fixed tissue, the method allows easy upscaling to obtain enough ChIP material for genome-wide profiling. Our protocol has been used successfully to map various DNA-binding proteins such as transcription factors, signaling mediators, components of the transcription machinery, chromatin modifiers and post-translational histone modifications, and for this to be done at various stages of embryogenesis. Lastly, this protocol should be widely applicable to other model and non-model organisms as more and more genome assemblies become available.

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

The question of how chromatin regulators and chromatin states affect the genome in vivo is key to our understanding of how early cell fate decisions are made in the developing embryo. ChIP-Seq&#8212;the most popular approach to investigate chromatin features at a global level&#8212;is outlined here for Xenopus embryos.

染色质监管机构和各国的全基因组快照<em&gt;爪</em&gt;通过胚胎的ChIP-SEQ

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and ...

科研

发育生物学

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 ...

遗传学

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

心脏分化的表观遗传调控胚胎干细胞和组织

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

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 from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

从人类胚胎干细胞的染色质免疫沉淀

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

生物学

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, ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

使用早期的小鼠胚胎组织特异性基因的染色质免疫沉淀检测

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

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

Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

Chromatin immunoprecipitation (ChIP) is a widely-used method for determining the interactions of different proteins with DNA in chromatin of living cells. Examples include sequence-specific DNA binding transcription factors, histones and their different modification states, enzymes such as RNA polymerases and ancillary factors, and DNA repair components. Despite its ubiquity, there is a lack of up-to-date, detailed methodologies for both bench preparation of material and for accurate analysis allowing quantitative metrics of interaction. Due to this lack of information, and also because, like any immunoprecipitation, conditions must be re-optimized for new sets of experimental conditions, the ChIP assay is susceptible to inaccurate or poorly quantitative results.
Our protocol is ultimately derived from seminal work on transcription factor:DNA interactions1,2 , but incorporates a number of improvements to sensitivity and reproducibility for difficult-to-obtain cell types. The protocol has been used successfully3,4 , both using qPCR to quantify DNA enrichment, or using a semi-quantitative variant of the below protocol.
This quantitative analysis of PCR-amplified material is performed computationally, and represents a limiting factor in the assay. Important controls and other considerations include the use of an isotype-matched antibody, as well as evaluation of a control region of genomic DNA, such as an intergenic region predicted not to be bound by the protein under study (or anticipated not to show changes under the experimental conditions). In addition, a standard curve of input material for every ChIP sample is used to derive absolute levels of enrichment in the experimental material. Use of standard curves helps to take into account differences between primer sets, regardless of how carefully they are designed, and also efficiency differences throughout the range of template concentrations for a single primer set. Our protocol is different from others that are available5-8 in that we extensively cover the later, analysis phase.

We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.

生物质的使用限制金额高效的染色质免疫沉淀

Chromatin immunoprecipitation (ChIP) is a  widely-used method for determining the interactions of different proteins with  DNA in chromatin of living ...

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 ...