我们感谢 IGBMC 高吞吐量测序设施的所有工作人员, 即 "法国 Génomique" 财团 (ANR10-INBS-09-08) 的成员和所有 IGBMC 一般事务人员, 特别是 IGBMC 动物设施的工作人员。这项工作得到了来自 CNRS、INSERM、AFM、甲国家组织勒癌症的赠款、法国全国基金在 Investissements 艾文莉标记 ANR-10-IDEX-0002-02、Labex INRT ANR-10-IDEX-0001-02 和ANR-AR2GR-16-CE11-009-01资助者在研究设计、数据收集和分析、决定出版或准备原稿方面没有作用。甲组织的支持下, 美国的 ANR-AR2GR-16-CE11-009-01, 和五, U 由 Ministère de Enseignement 等研究。ID 是一个 ' équipe labellisée ' 的甲国立组织勒癌症。

根据国家动物保育指导方针 (欧洲委员会指令 86/609/中欧和东欧), 按照有关实验室动物的护理和使用的机构准则, 对老鼠进行了保存;法国法令 87-848)。所有程序均由法国国家伦理委员会批准. 
 1. 肌肉组织的分离 <ol> <li> 由颈椎脱位牺牲一成人6至8周大鼠。Sterlise 用70% 乙醇冲洗四肢, 用细点剪刀和镊子解剖后肢肌肉 (腓肠肌、胫骨前、四头肌). 
	注意: 一只老鼠应该产生大约500毫克的组织. </li>
	<li> 将2毫升测试管中的肌肉剁碎, 其中含有1毫升的 ice-cold 低渗缓冲液, 以均匀制备小 (#60; 2-3 mm 3 ) 件使用细剪刀和离开管晃动在一个板凳顶部搅拌器在4和 #176; C 为 5-10 min. </li>
</ol> 2。组织裂解 注意: 对于所有的缓冲区组合, 请参阅 表 1 。
<ol> <li> 将匀浆转移到14毫升的圆底管和重5毫升的冷低渗缓冲液 (无 EDTA 蛋白酶抑制剂鸡尾酒和 PMSF). 
	<ol> <li> 使用松散的 dounce (3 分钟内 20-30 冲程) 或机械组织均质体在 15-三十年代的圆底14毫升管中必须碎肌肉组织. 
		注: 在这个阶段, 可以通过光显微镜对裂解的效率进行评估, 如果需要, 还可以进行额外的干扰. </li>
	</ol> </li> <li> 使用冷低渗缓冲将匀浆转移到15毫升管和填充体积到10毫升。在继续下一只鼠标之前, 按照下面的描述修复匀浆. </li>
</ol> 3。固定 <ol> <li> 将甲醛添加到1% 的最终浓度, 并在室温下摇动10分钟. </li>
	<li> 在每管中加入甘氨酸最终浓度为0.125 米, 以停止固定和摇动 #160; 5-10 分钟室温. </li>
</ol> 4。核准备工作 注意: 请参阅 表 1 中的所有缓冲区组合。
<ol> <li> 使用松散的 dounce (5-10 笔画) 必须固定的裂解产物. 
	<ol> <li> 传输到15毫升管和离心机在 1000 x g 5 分钟, 在4和 #176; C 获得细胞核和细胞碎片. </li>
	</ol> </li> <li> 在新鲜的5毫升低渗缓冲器中移除上清和重颗粒。通过70和 #181 将裂解液过滤到50毫升的管中。重新过滤滤液通过40和 #181; m 细胞过滤器. </li>
	<li> 将滤液转移到15毫升管和离心机在 1000 x g 5 分钟, 在4和 #176; C 以获得核颗粒. 
	注: 在这个阶段, 原子核可以立即声, 或在液氮中以干球的形式被冻结, 并储存在-80 和 #176; C. </li>
</ol> 5。超声 <ol> <li> 评估核颗粒的体积 (大约50和 #181; L 为机械和 dounce 化) 和重它在超声缓冲3到4倍包装的核容量和几种为 10-15 min 在4和 #176; C 使用 sonicator. 
	注: 染色质可按以下描述立即分析 (6) 或冷冻保存在-80 和 #176; C. </li>
</ol> 6。评估超声的染色质碎片大小 注意: 请参阅 表 1 中的所有缓冲区组合。
<ol> <li> de-crosslink, 取30和 #181; 1.5 毫升试管中的染色质, 并添加20和 #181; l 5 M 氯化钠。完成卷到500和 #181; L 和孵育在65和 #176; C 过夜. 
	<ol> <li> 第二天, 使用1和 #181 进行处理; l 蛋白酶 K (20 毫克/毫升), 10 和 #181; l 2 米, pH 6.8, 10 和 #181; 0.5 米 EDTA 为1小时, 在42和 #176; c. 执行苯酚-氯仿/氯仿萃取, 并沉淀的 DNA 1 量的 acetat 钠e (3 M) 和2容量100% 乙醇为一 h 在-80 和 #176; c 或隔夜在-20 和 #176; c. </li>
	</ol> </li> <li> 通过离心在 13500 x g 上进行15分钟的醒酒, 用冷70% 乙醇将颗粒彻底洗净, 再离心5分钟醒酒上清液并风干颗粒. </li>
	<li> 重原始卷 (30 和 #181; L) 中的颗粒。在 260 nm/280 纳米的光度法测定分光光度计中的 DNA 浓度。吸管 500-1000 ng dna 与 dna 大小梯在单独的车道上的1.5% 琼脂糖凝胶和执行电泳评估的片段大小染色质. 
	注意: 碎片大小应介于 200-500 碱基对之间, 并且不应有可检测到的高分子量片段对应于或不完整的 DNA。如果需要, 染色质溶液 (步骤 5) 可以 re-sonicated 较长时间, 然后重新如上所述, 直到获得最佳尺寸. </li>
</ol> 7。染色质沉淀 (芯片) 注意: 请参阅 表 1 中的所有缓冲区组合。
<ol> <li> 在乙醇中通过 aliquoting 1 毫升的50% 浆块将蛋白 G 琼脂珠阻止。在台式离心机中离心 400 x g, 用1毫升的 TE 缓冲离心机在 400 x 克上进行颗粒化, 然后重复洗涤. 
	<ol> <li> 在第二次离心后, re-suspend 在1毫升的芯片稀释缓冲器中, 25 和 #181; BSA (库存20毫克/毫升) 和20和 #181; l 酵母 tRNA (库存10毫克/毫升)。在4和 #176 中, 用旋转 (40-60 rpm) 将珠子至少阻挡在2小时前; C. </li>
		<li> 稀释50和 #181; g 的染色质 (步骤 5) 8 至10次使用芯片稀释缓冲器。添加50和 #181; 在4和 #176 中, 在2° c 旋转 (40-60 rpm) 上, 将被阻挡的珠浆和孵育的 L. 在400和 #176 上离心, 在 4 x g 处获得名册染色质并将其转移到新鲜的试管中. </li>
	</ol> </li> <li> 对于沉淀, 添加适当数量的主要抗体 (, 如 , 1-2 和 #181; g 每10和 #181; g), 并在夜间孵育4和 #176 的旋转; C. 
	注: 最佳抗体量可以由供应商推荐, 也可以通过执行不同数量的抗体的 qPCR 来确定, 直到最佳的富集被观察到。此外, 蛋白质 G 琼脂可能被蛋白质琼脂所取代, 这取决于所使用抗体的亚型. </li>
	<li> 添加被阻止的珠子第二天和孵化1小时与旋转 (40-60 rpm) 在4和 #176; c. 离心机在 400 x g 为 20-三十年代, 以颗粒的珠子, 清除上清, 并开始切屑洗. </li>
	<li> 芯片洗涤: 执行以下洗涤与缓冲: 一次以低盐缓冲, 然后两次与高盐缓冲, 两次与氯化缓冲, 和最后两次与 TE 缓冲. 
	<ol> <li> 执行每洗10分钟的旋转 (40-60 rpm), 在4和 #176; C 和颗粒的珠之间的每个洗涤的离心在 400 x g 为 20-三十年代在一个台式离心机. </li>
	</ol> </li> <li> 洗脱时, 删除最后的洗涤和重250和 #181 中的珠子; L 洗脱缓冲液 (新鲜准备) 在室温下15分钟, 在晃动时. </li>
	<li> 离心在 400 x g 和收集洗在一个新鲜的管。执行此步骤两次, 然后 de-crosslink 洗一夜20和 #181; l 氯化钠5米和1和 #181; 核糖核酸 A (10 毫克/毫升), 用蛋白酶 K 和苯酚-氯仿/氯仿机治疗如步骤6所示. </li>
	<li> 重在50和 #181 中, 根据标准的协议使用等分进行芯片 qPCR 15 检查芯片的质量. </li>
</ol>

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作者声明他们没有竞争的金融利益。

在这里, 我们描述了一个新的协议, 从成年小鼠骨骼肌肉制备染色质, 并表明, 这种染色质是适用于芯片实验, 检测转录因子结合和共价键组蛋白修饰肌纤维核。此协议涉及几个关键步骤。第一种是组织破坏, 可以通过 dounce 或机械剪切来进行。机械剪切速度快, 重现性好, 因此是选择的方法。不过, 如果没有合适的仪器, 也可以由 douncing, 在进行固定步骤之前, 建议在显微镜下对裂解效率进行评估。固定的全组织裂解, 而不是固定的孤立核, 允许更多的可再生超声在较小的体积和短期内。我们还选择在中断后修复裂解, 而不是在中断之前修复组织, 如托马斯et al.所描述的那样。10预修复组织导致化效率降低。该协议的基础上, 机械剪切也比其他建议的解决方案, 如胶原酶消化, 这涉及长期孵化的解剖组织在37° c8。在此期间, 基因表达和转录因子结合的变化可能发生。因此, 尽可能快地修复细胞核是更忠实地捕捉正常生理状态的关键。
第二个关键步骤是从固定的裂解液中提纯原子核。为此, 顺序过滤固定的裂解液, 首先通过70µm 细胞过滤器, 以消除较大的碎片, 然后通过一个40µm 细胞过滤器, 以消除罚款的碎片, 避免堵塞的滤器和最大的产量的核获得。经过滤纯化后, 细胞核声, de-crosslinking 后确定 DNA 片段大小。一个典型的准备从两个后肢的一只老鼠产生100µg 的染色质。将裂解从多达3只老鼠中收集起来, 可以通过减少制剂中的损失来提高产量。这一过程是有效的, 因为它允许恢复多达75% 的基因组 DNA 染色质 (数据没有显示)。
用于评估转录因子结合和后生修饰的芯片实验证明了该协议对于研究肌纤维细胞核基因调控的适用性。强大的和强健的芯片序列信号 ii 和 H3K27ac 是由染色质产生的, 允许识别肌肉纤维的身份基因由其独特的 H3K27ac 和政客 ii 配置文件7。与卫星或内皮细胞中表达的基因相比, 在纤维核中强烈表达的 H3K27ac 和 II. 类基因的含量更高, 这表明信号主要来自纤维核。这也证实了 Tead4 芯片信号的丢失使用染色质从老鼠那里它是特别地被灭活在肌肉纤维。然而, 对于诸如Vcam1、 Pax7 (未显示的数据) 等卫星单元标记, 较弱但明显高于背景信号的情况, 可以检测出这些细胞的染色质也存在。我们预计, 我们的协议应适用于其他技术, 使用甲醛固定染色质, 如3碳/4 c 和细胞核染色质构象捕获技术。使用我们的协议结合芯片序列的转录因子和染色质修饰与染色质构象捕获应该在未来允许详细了解的基因调控机制的肌肉纤维和如何调节这些可能或扰乱生理刺激, 如运动, 空腹, 高脂肪饮食, 失神经支配, 或在疾病中, 使用染色质制备的基因修饰小鼠复制疾病如 X 连锁中央肌病14。
总之, 这种低成本和时间的协议允许隔离的肌肉纤维核, 可以立即声或冻结在-80 ° c, 为不可告人的使用。使用该协议制备的染色质提供了第一个基因组宽的芯片序列数据从肌肉纤维7 , 并将促进未来的研究, 基因调控在这个组织。

染色质沉淀 (芯片) 结合定量聚合酶链反应 (qPCR) 和高通量测序 (芯片序列) 已成为选择的方法研究转录和表观遗传调控的基因表达在各种组织和单元格类型1。这项技术允许基因组范围内共价染色质修饰, 蛋白的变体占用, 和转录因子绑定2,3。
虽然从培养细胞中执行芯片是很好的建立, 从哺乳动物组织芯片仍然是更具挑战性。为芯片准备染色质涉及几个关键步骤, 需要优化每一个组织和细胞类型。染色质可以从细胞裂解培养细胞或在其细胞核纯化后制备。在哺乳动物组织的情况下, 有效的裂解, 甲醛固定和纯化的细胞核是至关重要的, 以确保最佳的恢复染色质。此外, 选择是否修复核之前或之后, 他们的纯化必须实验确定。尽管有这些障碍, 芯片已成功地从组织, 如肝脏, 睾丸, 或大脑4,5,6。在骨骼肌肉的情况下, 破坏这种身体上的抗性组织, 它表现出高含量的结构蛋白, 和孤立的细胞核是特别具有挑战性的。鉴于这种特异性, 为其他组织优化的协议不会给骨骼肌肉带来令人满意的结果。
在这里, 我们描述了一个协议, 隔离芯片级染色质从小鼠骨骼肌组织, 包括物理扰乱组织, 甲醛固定, 然后分离细胞核和超声。通过对各种转录因子、RNA 聚合酶 II 和共价组蛋白修饰的芯片 qPCR 和芯片序列进行了研究, 证明了该方法制备适合于该组织芯片的染色质的效率7。
这种新的技术比以前报告的协议8要快得多, 包括长时间胶原酶消化步骤, 在此期间, 转录因子基因组本地化的变化和基因表达的改变可能会发生。原子核被隔绝和固定的快速, 使这里描述的方法特别有吸引力准备染色质更加忠实地捕获本机基因组占有状态。此外, 虽然其他方法的染色质分离或蛋白质提取从肌肉组织已被描述8,9,10 , 并用于芯片 qPCR 实验选定的基因, 没有芯片 seq已报告使用这些数据。所报告的方法适用于转录因子和组蛋白修饰芯片序列, 因此也应适用于 3/4 c 或细胞核染色质构象捕获应用。

<table><tbody><tr><td>T 25 digital ULTRA-TURRAX</td><td>T 18 ULTRA-TURRAX</td><td>0009022800</td><td></td></tr><tr><td>PMSF</td><td>SIGMA-ALDRICH CHIMIE SARL</td><td>P7626-25G</td><td></td></tr><tr><td>Protease Inhibitor Cocktail-EDTA Free</td><td>Roche Diagnostics</td><td>11873580001</td><td></td></tr><tr><td>Protein G Sepharose beads</td><td>SIGMA-ALDRICH CHIMIE</td><td>P-3296</td><td></td></tr><tr><td>Proteinase K</td><td>SIGMA-ALDRICH CHIMIE</td><td>P-2308</td><td></td></tr><tr><td>Cell Strainers 70um</td><td>Corning BV</td><td>431751</td><td></td></tr><tr><td>Cell Strainers 40um</td><td>Corning BV</td><td>352340</td><td></td></tr><tr><td>Formaldehyde EM grade</td><td>Euromedex</td><td>15710-S</td><td></td></tr><tr><td>Rnase A</td><td>Fischer Scientific</td><td>12091039</td><td></td></tr><tr><td>Phenol:Chloroform:Isoamyl Alcohol 25:24:1</td><td>SIGMA-ALDRICH CHIMIE</td><td>P3803</td><td></td></tr><tr><td>BSA</td><td>SIGMA-ALDRICH CHIMIE</td><td>B4287-25G</td><td></td></tr><tr><td>yeast tRNA</td><td>SIGMA-ALDRICH CHIMIE</td><td>R5636</td><td></td></tr><tr><td>Glycogen Blue</td><td>AMIBION</td><td>AM9516</td><td></td></tr><tr><td>Pol II ChIP Antibody</td><td>Santa Cruz</td><td>SC-9001</td><td></td></tr><tr><td>H3K27ac ChIP Antibody</td><td>Active Motif</td><td>39133</td><td></td></tr><tr><td>Tead4 ChIP Antibody</td><td>Aviva Systems Biology</td><td>(ARP38276_P050)</td><td></td></tr><tr><td>IGEPAL</td><td>SIGMA-ALDRICH CHIMIE</td><td>I-3021</td><td></td></tr><tr><td>E220 Focused Ultrasonicator</td><td>Covaris E220</td><td></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;Additional items&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>Loose Dounce</td><td></td><td></td><td></td></tr><tr><td>15 ml and 50 ml falcon tubes</td><td></td><td></td><td></td></tr><tr><td>14 ml round bottom tubes</td><td></td><td></td><td></td></tr><tr><td>1.5 ml and 2 ml Eppendorf tubes</td><td></td><td></td><td></td></tr><tr><td>70 &amp;mu;m and 40 &amp;mu;m cell strainers (Corning)</td><td></td><td></td><td></td></tr></tbody></table>

improved protocol for chromatin immunoprecipitation from mouse skeletal muscle

我们描述了一个有效的和可复制的协议, 从成年小鼠骨骼肌的染色质制备, 一个具有高含量结构蛋白的身体抗性组织。从成年小鼠身上解剖肢体肌肉被机械化, 或切碎和 douncing 的组合, 在低渗缓冲液中, 在甲醛固定的细胞裂解。固定核通过机械化或 douncing 的进一步循环纯化, 并连续过滤去除细胞碎片。纯化后的核可以立即声或在冷冻后的后期进行。染色质可以有效地声, 适用于染色质沉淀实验, 如为转录因子、RNA 聚合酶 II 和共价键组蛋白修饰所获得的剖面所示。使用本协议所制备的染色质检测到的绑定事件主要是在肌纤维核中发生的, 尽管其他纤维相关的卫星和内皮细胞存在染色质。因此, 本议定书适用于研究成年小鼠骨骼肌的基因调控。

为了分离细胞核, 我们在15或四十五年代, 在1.8万和 2.2万 rpm 进行了解剖和碎肌组织的机械均质化 (参见材料表)。在所有条件下, 细胞核可以与组织的碎片分离, 但产量是最好的使用较低的速度 (图 1A-b)。原子核也可以由 douncing 为 3-5 min (图 1A, 和数据没有显示), 但机械均匀化是选择的方法, 因为最理想的产量的原子核可以实现在尽可能少的十五年代, 使时间的重要增益, 特别是如果必须处理多个示例 (图 1B)。使用该协议, 多达 2.7 x 107核从500毫克的组织 (相当于1鼠标的组织) 可以隔离, 以产生100µg 染色质。
为了优化染色质碎片从固定核, 我们声5至20分钟。在设置 (请参阅材料表) 下, 10 分钟超声是最佳生成染色质, 平均片段大小为 250 bp (图 2)。在考虑到所使用的 sonicator 类型的情况下, 应该对这一步骤进行优化试验。
为了评估上述协议所制备的肌肉染色质的质量, 对转录因子、组蛋白修饰或 RNA 聚合酶 ii (ii) 进行了芯片 qPCR 和芯片-seq 实验。qPCR 对糖皮质激素受体 (gr) 的存在或缺乏地塞米松 (德克斯) 治疗显示, 强德克依赖性 gr 丰富的调控元素的Redd1和Murf1基因, 已知是德克和 gr调节肌肉纤维11, 而在睾丸特定的Prm1启动子中没有出现信号, 用作负控制 (图 3A)。
qPCR 对乙酰赖氨酸27的组蛋白 H3 (H3K27ac), 在活性增强剂和促进剂中发现的共价修饰, 显示在肌纤维活性的蛋白 (Des) 启动子上的强富集, 与负基因控制区域 (图 3B)。H3K27ac 芯片-seq 在整个Des轨迹中显示了这个标记的高水平, 这是典型的 "super-enhancer" 调节组织识别基因12 (图 4A)。同样地, 在这个轨迹上, 二级芯片也显示出高水平的转录。
qPCR 转录因子 Tead4, 其中发挥重要作用, 在肌细胞分化13,7, 揭示了其丰富的前描述的绑定站点在Ccnd1和Ifrd1基因 (图 3C)。重要的是, 在肌肉纤维 (Tead4skm-)7 (图 3C) 中, 利用小鼠的染色质制备了 Tead4, 从而减少了浓缩。对 Tead1 和 Tead4 的芯片序列数据的分析显示, 它们绑定到Kdm5a基因启动子中的一个站点, 使用来自野生型肌肉的染色质, 而 Tead4 信号, 但不是 Tead1, H3K27ac, 或第二号, 是失去了使用染色质从Tead4skm小鼠的肌肉 (图 4B)。类似地, Tead1 和 Tead4 绑定到Adssl1基因下游的调节元素, 从野生型肌肉中看到染色质, 而 Tead4 绑定则是选择性地从Tead4skm-/肌肉中使用染色质丢失 (图 4C)。这些观察表明, 在芯片 seq 数据中看到的 Tead4 信号来自于肌肉纤维的结合。
为了确定与肌纤维相关的其他细胞类型对芯片 seq 结果的贡献, 我们评估了在肌肉卫星细胞和Pecam1中活跃的Vcam1基因中的 H3K27ac 和 II 信号, 这是一个标记血管内皮细胞能灌溉肌肉。与在Des或Adssl1基因中看到的高水平相比, 在Vcam1和Pecam1基因中看到的 H3K27ac 和 II 型的水平要低得多 (图 4A和图 4C)。与相关组织中表达的肌肉纤维表达基因的信号差异很大, 这表明, 使用上述协议制备的染色质芯片中的信号主要来源于肌肉纤维。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56504/56504fig1.jpg"> 
图 1: 从成年老鼠肌肉中提纯细胞核.(A)由机械化 (1.8万 rpm, 四十五年代) 和 dounce 均匀化 (30 strokes/3 分钟) 所获得的组织裂解, 用台盼蓝染色, 在数字显微镜下观察, 图像在20X 倍放大时被捕获。缩放条 = 100 µm. (B)在所述条件下制备的500毫克组织 (相当于1只老鼠) 获得的细胞核数, 1.8万和 2.2万 rpm 为15和四十五年代或3分钟的 douncing。误差线代表平均± S.E.M. 为 n = 3 只老鼠为机械均匀化, 并且 n = 3 只老鼠为 douncing。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56504/56504fig1large.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/56504/56504fig2.jpg"> 
图 2: 染色质碎片大小的验证.被隔绝的核被声 5, 10, 15 或 20 min 如表明。在 de-crosslinking 和纯化后, 用琼脂糖凝胶电泳对溴溴化物进行了 DNA 片段大小的评估。M 是 DNA 大小标记。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56504/56504fig2large.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/56504/56504fig3.jpg"> 
图 3: 用于芯片 qPCR 的染色质的使用.(A)小鼠受腹腔注射的车辆或地塞米松 (10 毫克/千克) 和1.5 小时后, 染色质是由后肢肌肉制备的。对 GR 靶基因的富集在芯片的Redd1和Murf1,相比, 负控制鱼精蛋白启动子 (Prm1), 表示为输入沉淀的%。(B)芯片用于 H3K27ac 和 IgG 控制抗体, 并在蛋白启动子上进行分析, 并与基因负控制区进行比较。(C) Tead4 芯片是对从野生类型和肌肉特异性 Tead4 击倒小鼠 (Tead4skm-) 获得的肌肉染色质进行的, 并在Tead4和Ccnd1启动子中的 Ifrd1 绑定站点上进行分析, 与控制基因区域。在所有的实验中, 3 只小鼠的染色质被汇集, 误差条代表了三技术复制的学生 T 检验中的平均±东南亚 M。P 值和 #60; 0.001 **, #60; 0.0001 **;NS, 显著。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56504/56504fig3large.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/56504/56504fig4.jpg"> 
图 4: 肌纤维染色质的基因组图谱.(A)大学的截图加利福尼亚州的 y 在圣克鲁斯基因组浏览器的指示位点说明了高得多的 H3K27ac 和第二类蛋白基因, 高表达的肌肉纤维, 与Vcam1和Pecam1基因表达的卫星细胞和内皮细胞, 分别。(B) UCSC 的屏幕截图, 表明 Tead1 和 Tead4 的结合, 以及 H3K27ac 和油料 II, 在染色质从野生型 (重量) 肌肉相比, 从 Tead4skm/- (MT) 肌肉, 选择性失去 Tead4 结合是观察.在 GEO 数据库中, 可作为 GSE82193 提供芯片 seq 数据。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56504/56504fig4large.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>缓冲区名称</td>
			<td colspan="3">组成</td>
		</tr>
		<tr>
			<td rowspan="6">低渗缓冲器</td>
			<td colspan="3">10毫米 HEPES-KOH (pH 7.3)</td>
		</tr>
		<tr>
			<td colspan="3">10毫米氯化钾</td>
		</tr>
		<tr>
			<td colspan="3">5 mM 氯化镁2</td>
		</tr>
		<tr>
			<td colspan="3">0.1% NP-40</td>
		</tr>
		<tr>
			<td colspan="3">0.1 毫米 PMSF (新鲜地增加)</td>
		</tr>
		<tr>
			<td colspan="3">蛋白酶抑制剂鸡尾酒 (PIC) 1x (新鲜添加)</td>
		</tr>
		<tr>
			<td rowspan="8">超声缓冲区</td>
			<td colspan="3">1% SDS</td>
		</tr>
		<tr>
			<td colspan="3">10毫米 EDTA</td>
		</tr>
		<tr>
			<td colspan="3">20毫米三盐酸盐 pH 值8</td>
		</tr>
		<tr>
			<td colspan="3">150毫米氯化钠</td>
		</tr>
		<tr>
			<td colspan="3">0.1% 胆酸钠</td>
		</tr>
		<tr>
			<td colspan="3">1% 海卫 X-100</td>
		</tr>
		<tr>
			<td colspan="3">新鲜增加的0.1 毫米 PMSF</td>
		</tr>
		<tr>
			<td colspan="3">PIC 1x</td>
		</tr>
		<tr>
			<td rowspan="7">芯片稀释缓冲器</td>
			<td colspan="3">0.01% SDS</td>
		</tr>
		<tr>
			<td colspan="3">1.1% 海卫 X-100</td>
		</tr>
		<tr>
			<td colspan="3">1.2 毫米 EDTA</td>
		</tr>
		<tr>
			<td colspan="3">16.7 毫米三盐酸 pH8</td>
		</tr>
		<tr>
			<td colspan="3">167毫米氯化钠</td>
		</tr>
		<tr>
			<td colspan="3">新鲜增加的0.1 毫米 PMSF</td>
		</tr>
		<tr>
			<td colspan="3">PIC 1x</td>
		</tr>
		<tr>
			<td rowspan="5">低盐缓冲器</td>
			<td colspan="3">0.1% SDS</td>
		</tr>
		<tr>
			<td colspan="3">1% 海卫 X-100</td>
		</tr>
		<tr>
			<td colspan="3">500毫米 EDTA</td>
		</tr>
		<tr>
			<td colspan="3">20毫米三盐酸 pH8</td>
		</tr>
		<tr>
			<td colspan="3">150毫米氯化钠</td>
		</tr>
		<tr>
			<td rowspan="5">高盐缓冲器</td>
			<td colspan="3">0.1% SDS</td>
		</tr>
		<tr>
			<td colspan="3">1% 海卫 X-100</td>
		</tr>
		<tr>
			<td colspan="3">500毫米 EDTA</td>
		</tr>
		<tr>
			<td colspan="3">20毫米三盐酸 pH8</td>
		</tr>
		<tr>
			<td colspan="3">500毫米氯化钠</td>
		</tr>
		<tr>
			<td rowspan="4">氯化缓冲区</td>
			<td colspan="3">250毫米氯化</td>
		</tr>
		<tr>
			<td colspan="3">1% NP-40</td>
		</tr>
		<tr>
			<td colspan="3">1毫米 EDTA</td>
		</tr>
		<tr>
			<td colspan="3">10毫米三盐酸盐 pH 值8</td>
		</tr>
		<tr>
			<td rowspan="2">洗脱缓冲器</td>
			<td colspan="3">1% SDS</td>
		</tr>
		<tr>
			<td colspan="3">100 mM NaHCO3</td>
		</tr>
		<tr>
			<td>三 EDTA (TE) 缓冲器</td>
			<td colspan="3">50毫米三盐酸盐 pH 值8</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="3">1毫米 EDTA</td>
		</tr>
	</tbody>
</table>
表 1: 缓冲组合。

Watch this Scientific Journal Video about Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle at JoVE.com

Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle

改进的小鼠骨骼肌染色质沉淀的协议

本文介绍了一种新的制备成年小鼠骨骼肌染色质的方法, 该方法适合于用染色质沉淀研究肌纤维的基因调控。

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a ...

improved-protocol-for-chromatin-immunoprecipitation-from-mouse

Nanyang Technological University

Research

JoVE Journal

Biochemistry

8.5K 次观看.  Institut de Génétique et de Biologie Moléculaire et Cellulaire. 本文介绍了一种新的制备成年小鼠骨骼肌染色质的方法, 该方法适合于用染色质沉淀研究肌纤维的基因调控。

视频: Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle

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 ...

科研

遗传学

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

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Here, we describe a chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation protocol to generate global epigenomic profiles from low-abundance chicken embryonic samples.

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, ...

A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a challenge for ChIP due to technical difficulty in isolation of high-quality nuclei with minimal contamination of myofibrils. Previous protocols have attempted to purify nuclei before cross-linking, which incurs the risk of altered DNA-protein interaction during the prolonged nuclei preparation process. In the current protocol, we first cross-linked the skeletal muscle tissue collected from mice, and the tissues were minced and sonicated. Since we found that ultracentrifugation was not able to separate nuclei from myofibrils using cross-linked muscle tissue, we devised a sequential filtration procedure to obtain high-quality nuclei devoid of significant myofibril contamination. We subsequently prepared chromatin by using an ultrasonicator, and ChIP assays with anti-BMAL1 antibody revealed robust circadian binding pattern of BMAL1 to target gene promoters. This filtration protocol constitutes an easily applicable method to isolate high-quality nuclei from cross-linked skeletal muscle tissue, allowing consistent sample processing for circadian and other time-sensitive studies. In combination with next-generation sequencing (NGS), our method can be deployed for various mechanistic and genomic studies focusing on skeletal muscle function.

We present a filtration-based protocol to isolate high-quality nuclei from cross-linked mouse skeletal muscle wherein we removed the need for ultracentrifugation, making it easily applicable. We show that chromatin prepared from the nuclei is suitable for chromatin immunoprecipitation and likely chromatin immunoprecipitation sequencing studies.

从交联骨骼肌制备高质量核的基于过滤的方法进行染色质免疫沉淀

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a ...

生物化学

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 ...

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 ...

从小鼠肢体肌肉中分离卫星细胞和流式细胞术分析

An Efficient Protocol for CUT&amp;RUN Analysis of FACS-Isolated Mouse Satellite Cells

Genome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient protocol for the fluorescence-activated cell sorting (FACS) isolation of satellite cells from the limb muscle, a tissue with a high content of structural proteins. Dissected limb muscles from adult mice were mechanically disrupted by mincing in medium supplemented with dispase and type I collagenase. Upon digestion,&#160;the homogenate was filtered through cell strainers, and cells were suspended in FACS buffer. Viability was determined with fixable viability stain, and immunostained satellite cells were isolated by FACS. Cells were lysed with Triton X-100 and released nuclei were bound to concanavalin A magnetic beads. Nucleus/bead complexes were incubated with antibodies against the transcription factor or histone modifications of interest. After washes, nucleus/bead complexes were incubated with protein A-micrococcal nuclease, and chromatin cleavage was initiated with CaCl2. After DNA extraction, libraries were generated and sequenced, and the profiles for genome-wide transcription factor binding and covalent histone modifications were obtained by bioinformatic analysis. The peaks obtained for the various histone marks showed that the binding events were specific for satellite cells. Moreover, known motif analysis unveiled that the transcription factor was bound to chromatin via its cognate response element. This protocol is therefore adapted to study gene regulation in adult mice limb muscle satellite cells.

作者聚焦:小鼠肌肉干细胞中的 Cistrome 分析 

Presented here is an efficient protocol for the fluorescence-activated cell sorting (FACS) isolation of mouse limb muscle satellite cells adapted to the study of transcription regulation in muscle fibers by cleavage under targets and release using nuclease (CUT&amp;RUN).

一种用于FACS分离的小鼠卫星细胞的CUT&amp;RUN分析的有效方案

Genome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient ...

发育生物学

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 ...

改进的小鼠骨骼肌染色质沉淀的协议

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染色质免疫沉淀（ChIP）在小鼠T细胞系

低丰度胚胎标本的染色质沉淀 (芯片) 协议

从交联骨骼肌制备高质量核的基于过滤的方法进行染色质免疫沉淀

从酿酒酵母中组蛋白修饰的染色质沉淀 (芯片)

小鼠棕色脂肪组织的染色质免疫沉淀

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

一种用于FACS分离的小鼠卫星细胞的CUT&RUN分析的有效方案

一种用于FACS分离的小鼠卫星细胞的CUT&RUN分析的有效方案

一种用于FACS分离的小鼠卫星细胞的CUT&RUN分析的有效方案

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