我们非常感谢P.Käse和T. Schmidt-Wöll的优秀技术援助。这项工作得到了德国研究基金会（DFG）（德国研究基金会），马克斯·普朗克社会和弗莱堡的EXC 294的合作研究资助TRR81和海森堡计划（BO 1639 / 5-1）以及心肺系统（ECCPS）在吉森至TB

注意: 表1中列出了本协议中使用的所有缓冲区和介质。 第1天和第2天交换单元格<ol><li>将小鼠T细胞从6孔板转移到50mL管中，并在4℃和〜271×g离心5分钟。将细胞沉淀重悬于30mL的IMDM细胞培养基中，并使用Neubauer室计数细胞。 </li><li>将20×10 6个细胞的等分试样收集到新的50mL管中。 </li><li>将甲醛（FMA）直接加入到细胞培养基中1％终浓度，并在室温下孵育10分钟。 注意:如果吸入，FMA有毒，因此在通风橱下工作并佩戴适当的保护设备。另外，请按照主办机构的规定处理FMA废物。 </li><li>加入1/8体积的1M甘氨酸，pH7.0，并在室温下孵育5分钟。 </li><li>通过离心沉淀5次细胞分钟在4℃和〜271×g，并用10mL磷酸盐缓冲盐水（PBS）洗涤。 </li><li>用1 mL PBS洗涤细胞沉淀，并转移至1.5 mL管。 </li></ol>细胞裂解和染色质剪切<ol><li>将细胞沉淀重悬于1mL SDS裂解缓冲液中，并在冰上孵育10分钟。 </li><li>将每个样品转移到超声处理管中，并使用聚焦超声波仪根据表2所示的设置进行剪切。 </li><li>剪切后，将样品转移至1.5 mL试管，并在4°C和约18,000 x g离心10 min。 </li><li>将上清液转移到新管中。 </li><li>收集50μL，根据步骤3确定剪切效率，并在液氮中快速冷冻剪切的裂解物。将溶胞产物在-80℃下储存在一次性等分试样中。 </li></ol> 3.剪切效率的测定<ol><li>加入50μL洗脱缓冲液至50＃181; L每个剪切裂解物，并在65℃下孵育过夜，摇动。 </li><li>加入100μLTE缓冲液和4μL10 mg / mL RNA酶A（0.2μg/μL终浓度）。混合并在37℃下振荡孵育2小时。 </li><li>加入2μL20mg / mL蛋白酶K（0.2μg/μL终浓度）至每个样品，并在55℃下摇动孵育2小时。 </li><li>将每个样品转移到适用于苯酚/氯仿/异戊醇萃取的凝胶管中;按照制造商的说明处理。 </li><li> 加入200μL苯酚/氯仿/异戊醇（25:24:1），摇匀管。在24℃和约16,000xg离心5分钟，并将上层转移到新管中。 注意事项:苯酚，氯仿，异戊醇吸入有腐蚀性;在通风橱下工作，并穿戴适当的个人防护装备。另外，处理苯酚，氯仿，异戊醇等遵守主办机构的规定。 <ol><li>重复一次</li></ol></li><li> 根据制造商的说明，使用纯化柱纯化DNA，并进行以下修改: <ol><li>洗膜两次。最后一次洗涤后，将管在室温下放置2分钟以蒸发剩余的乙醇。 </li><li>为了洗脱，加入50μLH 2 O，室温孵育1 min，旋转管1秒，以适当地浸湿膜，并在室温下孵育1分钟，然后通过离心1分钟洗脱DNA 24°C和〜17,900 x g。 </li></ol></li><li>使用高灵敏度试剂盒根据制造商的说明书在荧光计上定量纯化的DNA。 </li><li>使用高灵敏度试剂盒，在1.8％琼脂糖凝胶上分析约500ng纯化的DNA，并在电泳系统上分析约1ng。 注意:预期的DNA片段的e为200〜500bp。 </li></ol>第3天和第4天 4.免疫沉淀<ol><li>稀释1体积的剪切裂解物与5体积的稀释缓冲液。 </li><li>与30μL/ mL蛋白A预先准确的琼脂糖珠（根据附录A制备）在冷室中旋转30分钟。 </li><li>在约750 x g下，4℃离心5分钟。 </li><li>收集10％的输入并将其存储在4°C。 </li><li>将所需量的裂解液分成新的管（ 详见表3 ）。向每个管中加入所需的抗体（ 详见表3 ），并在4°C下旋转过夜。 </li><li>加入40μL蛋白A珠，并在4℃下旋转孵育1小时。 </li><li>用1mL低盐缓冲液，高盐缓冲液，LiCl缓冲液和TE缓冲液洗涤珠子（每次洗涤，在4℃下孵育5分钟，同时旋转和离心在4℃和约950×g下3分钟）。 详见表3 。 </li><li>加入110μL洗脱缓冲液，室温孵育15分钟，每2分钟涡旋。 </li><li>在4℃和约950×g离心3分钟，并将100μL上清转移到新的1.5-mL管中。 </li><li>用100μL洗脱缓冲液重复步骤4.7-4.8，并结合洗脱液。加入高达200μL的洗脱缓冲液输入样品。 </li><li>将所有样品在65℃下振荡孵育过夜。 </li></ol>第5天 5. DNA纯化<ol><li>向每个洗脱液中加入200μLTE缓冲液，加入8μL10 mg / mL RNA酶A（最终浓度为0.2μg/μL）。混合并在37℃下振荡孵育2小时。 </li><li>向每个洗脱液中加入4μL20 mg / mL蛋白酶K（0.2μg/μL终浓度），并在55°C振荡孵育2 h。 </li><li>将每个样品转移到凝胶管适用于苯酚/氯仿/异戊醇提取;按照制造商的说明处理。 </li><li> 加入400μL苯酚/氯仿/异戊醇（25:24:1），摇匀管。在24°C离心5 min，约16,000 x g。将上层转移到新管。 注意事项:苯酚，氯仿，异戊醇吸入有腐蚀性;在通风橱下工作，并穿戴适当的个人防护装备。另外，按照主办机构的规定处理苯酚，氯仿，异戊醇等废物。 <ol><li>重复一次</li></ol></li><li>根据制造商的说明，使用纯化柱纯化DNA，并进行以下修改: </li><li>洗膜两次。最后一次洗涤后，将管在室温下放置2分钟以蒸发剩余的乙醇。 </li><li>为了洗脱，加入50μL的H 2 O，在室温下孵育重复1分钟，旋转管1秒以适当地润湿膜，并在室温下孵育1分钟，然后在24℃下离心1分钟至约17,900×g。 </li></ol>

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作者没有竞争的经济利益。

ChIP是一种有效的技术，用于调查蛋白质或其PTM是否在特定基因组区域富集。由于生物或技术原因，ChIP测定结果通常具有挑战性。生物学原因有很多，包括蛋白质与DNA的弱或间接结合。还存在技术限制，例如有限的抗体特异性和低效细胞裂解或染色质剪切。故障排除指南（ 表5 ）可帮助读者解决ChIP测定可能遇到的问题。 该协议利用冷却聚焦超声波装置，可以有效地剪切成熟的小鼠T细胞系的染色质。协议的主要关键步骤由染色质的剪切表示，染色质必须始终针对每种细胞类型进行优化。建议改变细胞数量和超声处理循环次数。而且，她<html>样品中SDS沉淀物的存在会对培养效率产生负面影响，SDS沉淀缓冲液可能在冰上裂解细胞时形成。在这种情况下，最好在室温下裂解后样品孵育数分钟以减少SDS沉淀物的存在。建议优化方案以获得200至500 bp之间的剪切片段，并在进行免疫沉淀前始终评估样品的剪切质量。此外，必须针对每种抗体优化注射时间，抗体和/或细胞的量以及洗涤条件。 使ChIP程序成功的另一个关键步骤是抗体的选择，因为低特异性抗体显着降低免疫沉淀的效率。以前，统一的质量检测程序允许评估市场上可获得的几种抗体的特异性<html> ss ="xref"&gt; 19。筛选管道基于斑点印迹，Western印迹和ChIP，提供了适用于ChIP 19的高品质试剂列表。最近，使用高密度组蛋白肽微阵列平台评估了几种市售抗体的特异性，允许建立抗体特异性数据库20 。读者可以使用这个有用的工具来识别可用于ChIP实验的最具体的抗体。 该协议尚未对原始T细胞进行测试，在这种情况下，读者应参考在主要T细胞3，4，21上成功执行ChIP的其他已发布协议。值得注意的是，该方案已经成功地用于在小鼠祖细胞T细胞系以及小鼠内皮细胞系上进行ChIP。 ove_content"&gt; 5×10 6个细胞应用于每次免疫沉淀用于组蛋白标记的分析，因为该方案也可以合适，通过一些优化，在TF或辅因子上进行ChIP，最好增加细胞数量用于这些情况下的免疫沉淀。为了达到每个实验所需数量的细胞，在稀释稀释缓冲液中的染色质稀释之前，可以将更多等分的剪切裂解物合并在一起。 也可以修改该方案以对不直接与DNA结合的内源蛋白进行ChIP。在这种情况下，可能需要预先固定蛋白质 - 蛋白质交联剂（ 例如，二甲基己二亚胺酯，DMA）（有关更多信息，请参阅附录B ）。先前通过过表达与生物素标签融合的蛋白质来实现ChIP对辅因子的成功表现。在这种情况下，用链霉抗生物素蛋白 - 缀合物纯化蛋白质22进行了预先固定的DMA。

发育，分化和体内平衡取决于通过调节染色质结构的信号事件建立的特定基因表达程序，从而确定特定基因是否以细胞和时间特异性方式被激活或抑制。在T细胞发育期间，必须建立特异性基因表达程序，以正确地确定T细胞前体从双负（DN）到单阳性（SP）状态的成熟，通过几个中间阶段1 。几个实验室2,3,4,5,6在前几年广泛研究了T细胞发育过程中基因表达程序的动态调控。 通过组蛋白的翻译后修饰（PTM），交换具有组蛋白变体，核小体迁移和DNA甲基化的规范组蛋白调节基因的ON / OFF转换。几组已经研究了PTM和组蛋白变体的全基因组分布，以确定在近端和远端调节区7,8,9,10处与不同染色质状态相关的标记。信号级联通过在特异性增强子元件交换正染色质修饰酶（也称为染色质修饰剂）来编排动态染色质调节。这些染色质调节剂通过例如动态组蛋白甲基化和乙酰化来调节染色质结构，从而调节转录产物。 Notch信号通路11,12,13 ，14。 动态组蛋白PTMs;与组蛋白变体交换;并且可以通过染色质免疫沉淀（ChIP）测定来研究组蛋白，转录因子和辅因子的动态占据。使用高度特异性的抗体来纯化特定的DNA-蛋白复合物，通过定量PCR（qPCR）分析纯化的DNA。深度测序（ChIP-Seq）;或者现在不太频繁地与微阵列杂交（ChIP-ChIP）。 由于细胞的裂解，染色质的剪切和/或抗体的低特异性的并发症，ChIP测定有时是有挑战性的。已经采用了几种策略来改进协议，就像NEXSON 15一样 。使用冷却水浴超声波器避免样品加热，这可能会损害正在研究的蛋白质上存在的表位，但剪切样品所需的能量分散在水中。随着防止能量分散的聚焦超声波装置的发展，进一步改进。因此，聚焦超声波设备允许改善细胞裂解和染色质剪切，消除操作者诱导的变异并显着增加再现性。 该工作描述了一种协议（ 图1中的示意图 ），用于有效地在称为E2-10HA 16,17的成熟小鼠T细胞系中执行组蛋白的ChIP。 T细胞通常难以溶解，染色质的剪切已被揭示为低效率。在使用冷却聚焦超声波器的该方案中，针对E2-10HA小鼠T细胞系优化了细胞数量，裂解缓冲液和剪切设置。该协议允许人们在合理的时间内以高重现性执行ChIP。事实上，它需要大约两天剪切染色质并评估剪切的质量，三天进行免疫沉淀，逆转交联并纯化DNA。

<table><tbody><tr><td>Agilent High Sensitivity D5000 Reagents</td><td>Agilent Technologies</td><td>5067-5593</td><td></td></tr><tr><td>Agilent High Sensitivity D5000 ScreenTape</td><td>Agilent Technologies</td><td>5067-5592</td><td></td></tr><tr><td>Agilent Tapestation 4200</td><td>Agilent Technologies</td><td>G2991AA</td><td></td></tr><tr><td>Covaris S220 AFA System</td><td>Covaris</td><td>500217</td><td></td></tr><tr><td>dimethyl adipimidate (DMA)</td><td>Thermo Fisher Scientific</td><td>20660</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Pan-Biotech</td><td>1502-P121301</td><td></td></tr><tr><td>Formaldehyde (FMA)</td><td>Sigma-Aldrich</td><td>F8775</td><td></td></tr><tr><td>Insulin solution human&amp;nbsp;</td><td>Sigma-Aldrich</td><td>I9278-5ML</td><td></td></tr><tr><td>Iscove&#039;s modified Dulbecco&#039;s medium (IMDM)</td><td>Gibco</td><td>21980-032</td><td></td></tr><tr><td>Minimum essential medium non-essential amino acids (MEM NEAA)</td><td>Gibco</td><td>11140-035</td><td></td></tr><tr><td>nProtein A Sepharose 4 Fast Flow</td><td>GE Healthcare</td><td>17-5280-02</td><td></td></tr><tr><td>Penicillin-Streptomycin&lt;br /&gt;(10,000 U/mL)</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Peptone primatone</td><td>Sigma-Aldrich</td><td>P4963-100G</td><td></td></tr><tr><td>Phase Lock Gel Heavy 2&amp;nbsp;mL</td><td>5 Prime</td><td>2302830</td><td></td></tr><tr><td>Phenol/chloroform/isoamyl alcohol (25:24:1)</td><td>Roth</td><td>A156.2</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>GIBCO</td><td>14190-094</td><td></td></tr><tr><td>Proteinase K Solution (20 mg/mL), RNA grade</td><td>Thermo Fisher Scientific</td><td>25530049</td><td></td></tr><tr><td>QIAquick PCR Purification Kit (250)</td><td>Qiagen</td><td>28106</td><td></td></tr><tr><td>Qubit 3.0 Fluorometer</td><td>Thermo Fisher Scientific</td><td>Q33216</td><td></td></tr><tr><td>Qubit assay tubes</td><td>Thermo Fisher Scientific</td><td>Q32856</td><td></td></tr><tr><td>Qubit dsDNA HS Assay Kit</td><td>Thermo Fisher Scientific</td><td>Q32854</td><td></td></tr><tr><td>RNase A, DNase and protease-free (10&amp;nbsp;mg/mL)</td><td>Thermo Fisher Scientific</td><td>EN0531</td><td></td></tr><tr><td>Tube AFA Fiber &amp;amp; Cap</td><td>Covaris</td><td>520081</td><td></td></tr></tbody></table>

chromatin immunoprecipitation (chip) in mouse t-cell lines

信号途径通过调节不同水平的染色质结构来调节基因表达程序，例如通过组蛋白尾的翻译后修饰（PTM），组蛋白变体的规范组蛋白的交换和核小体驱逐。这种调节需要在定义为增强子的调节元件上招募染色质修饰酶的信号敏感转录因子（TF）的结合。了解信号级联如何调节增强子活性需要对TFs，染色质修饰酶的结合以及特定组蛋白标记和组蛋白变体的占据进行综合分析。染色质免疫沉淀（ChIP）测定利用高度特异性的抗体免疫沉淀特异性蛋白质/ DNA复合物。纯化的DNA的随后分析允许鉴定由抗体识别的蛋白质占据的区域。这个工作描述了一个有效率的协议组蛋白在成熟小鼠T细胞系中的ChIP ChIP。所提出的方案允许在合理的时间范围内以高重现性进行ChIP测定。

对成熟的小鼠T细胞进行ChIP分析，研究赖氨酸4组蛋白3（H3K4me1）的组蛋白标记单甲基化，赖氨酸4在组蛋白3（H3K4me3）上的三甲基化）和赖氨酸27乙酰化的富集组蛋白3（H3K27ac）以及核小体占有率，如panH3 ChIP所揭示的。通过1.8％琼脂糖凝胶（ 图2A ）和电泳系统（ 图2B ）的分析来评估剪切质量。 H3K4me1和H3K4me3通常用于分别鉴别增强子位点和启动子（ 图3A ）。事实上，H3K4me3在启动子5,8,14中是突出的，但不完全是富集的。活性增强剂的特征是H3K4me1和H3K27ac高度富集，H3K4me3（&lt;强力类="xfig"&gt;图3A，上图），而无活性的增强子呈现低水平的H3K4me3和H3K27ac但高水平的H3K4me1（ 图3A ，下图）。 如图3B所示 ， 甘油醛-3-磷酸脱氢酶 （ Gapdh ）基因是活性基因启动子 （Gapdh 启动子 ）的分析的代表。实际上H3K4me3和H3K27ac水平较高，H3K4me1水平较低。在图3B中 ， Deltex1 （ Dtx1 ）代表无活性增强子 （ Dtx1增强子 ），因为它对H3K4me1是高度富集的，并且H3K4me3和H3K27ac的富集性差。 基因沙漠是缺乏编码基因的区域的代表，通常被称为活性染色质标记的阴性对照。观察到H3K4me1是一个被广泛接受的增强子标记4,5 ， </sup&gt; 7，14，18，在基因沙漠不富集，表明该地区缺乏增强剂。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55907/55907fig1.jpg" /> 图1:提出的ChIP协议示意图。 <a href="http://ecsource.jove.com/files/ftp_upload/55907/55907fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55907/55907fig2.jpg" /> 图2:成熟小鼠T细胞系的剪切质量控制。 （ A ）剪切成熟小鼠T细胞系E2-10HA的两个不同等分试样，在1.8％琼脂糖凝胶上分析大约500ng纯化的DNA，以评价其剪切质量。 （ B ）通过电泳分析1ng来自样品2的纯化DNA以评价其剪切质量。 <a href="http://ecsource.jove.com/files/ftp_upload/55907/55907fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55907/55907fig3.jpg" /> 图3:表征增强子和启动子的染色质标记。 （ A ）活性增强剂（上图）的特征在于高H3K4me1，低H3K4me3和高H3K27ac，而无活性增强子（下图）具有高H3K4me1，低H3K4me3和低H3K27ac的特征。 （ B ） 如图2A所示的样品汇集在一起​​，用于ChIP分析与组蛋白标记H3K4me1，H3K4me3和H3K27ac以及组蛋白占用，如PanH3 ChIP所示。这个分析显示，持家基因的e启动子甘油醛-3-磷酸脱氢酶 （ Gapdh 启动子 ）对H3K4me3和H3K27ac高度富集，并且H3K4me1低富集。另一方面，无活性基因Deltex1 （ Dtx1增强子 ）的增强子对H3K4me1高度富集，但对于H3K4me3和H3K27ac不富集。使用panH3抗体的ChIP来研究核小体占有率。 基因沙漠被用作阴性对照。显示了一个代表性的实验。 <a href="http://ecsource.jove.com/files/ftp_upload/55907/55907fig3large.jpg" target="_blank">请点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> 试剂 </td><td> 最终集中 </td></tr><tr><td rowspan="5">稀释缓冲液</td><td>十二烷基钠硫酸基（SDS） </td><td> 0.01％（w / v） </td></tr><tr><td> Triton X-100 </td><td> 1.1％（v / v） </td></tr><tr><td>乙二胺四乙酸（EDTA）pH 8.0 </td><td> 1.2mM </td></tr><tr><td> Tris-HCl pH 8.1 </td><td> 16.7mM </td></tr><tr><td>氯化钠（NaCl） </td><td> 167mM </td></tr><tr><td rowspan="2"> DMA解决方案</td><td>己二酸二甲酯（DMA） </td><td> 10 mM </td></tr><tr><td>磷酸盐缓冲盐水（PBS） </td><td> 1X </td></tr><tr><td rowspan="3">洗脱缓冲液</td><td>十二烷基硫酸钠（SDS） </td><td> 1％（w / v） </td></tr><tr><td>乙二胺四乙酸（EDTA）pH 8.0 </td><td> 10 mM </td></tr><tr><td> Tris-HCl pH 8.0 </td><td> 50 mM </td></tr><tr><td rowspan="5">高盐缓冲液</td><td>十二烷基硫酸钠（SDS） </td><td> 0.1％（w / v） </td></tr><tr><td> Triton X-100 </td><td> 1％（v / v） </td></tr><tr><td> Ethylenediami净四乙酸（EDTA）pH 8.0 </td><td> 2mM </td></tr><tr><td> Tris-HCl pH 8.1 </td><td> 20 mM </td></tr><tr><td>氯化钠（NaCl） </td><td> 500 mM </td></tr><tr><td rowspan="6"> IMDM介质</td><td> Iscove修改后的Dulbecco介质（IMDM） </td><td> 1X </td></tr><tr><td>胎牛血清（FBS） </td><td> 2％（v / v） </td></tr><tr><td>青霉素/链霉素</td><td> 1X </td></tr><tr><td>蛋白胨primatone </td><td> 0.3 mg / mL </td></tr><tr><td>胰岛素溶液人</td><td> 4.8 mg / mL </td></tr><tr><td>最低必需培养基非必需氨基酸（MEM NEAA） </td><td> 1X </td></tr><tr><td rowspan="4"> LiCl盐缓冲液</td><td>氯化锂（LiCl） </td><td> 0.25 M </td></tr><tr><td> IGEPAL-CA630 </td><td> 1％（v / v） </td></tr><tr><td>乙二胺四乙酸（EDTA）pH 8.0 </td><td> 1mM </td></tr><tr><td> Tris-HCl pH 8。1 </td><td> 10 mM </td></tr><tr><td rowspan="5">低盐缓冲液</td><td>十二烷基硫酸钠（SDS） </td><td> 0.1％（w / v） </td></tr><tr><td> Triton X-100 </td><td> 1％（v / v） </td></tr><tr><td>乙二胺四乙酸（EDTA）pH 8.0 </td><td> 2mM </td></tr><tr><td> Tris-HCl pH 8.1 </td><td> 20 mM </td></tr><tr><td>氯化钠（NaCl） </td><td> 150 mM </td></tr><tr><td rowspan="5">蛋白A琼脂糖珠洗涤缓冲液</td><td> Tris-HCl pH 8.0 </td><td> 20 mM </td></tr><tr><td>氯化钠（NaCl） </td><td> 500 mM </td></tr><tr><td>乙二胺四乙酸（EDTA）pH 8.0 </td><td> 2mM </td></tr><tr><td>十二烷基硫酸钠（SDS） </td><td> 0.1％（w / v） </td></tr><tr><td> IGEPAL-CA630 </td><td> 1％（v / v） </td></tr><tr><td rowspan="3"> SDS裂解缓冲液</td><td>十二烷基硫酸钠（SDS） </td><td> 1％（w / v） </td></tr><tr><td>乙二胺胺四乙酸（EDTA）pH 8.0 </td><td> 10 mM </td></tr><tr><td> Tris-HCl pH 8.1 </td><td> 50 mM </td></tr><tr><td rowspan="2"> TE缓冲区</td><td> Tris-HCl pH 8.0 </td><td> 10 mM </td></tr><tr><td>乙二胺四乙酸（EDTA）pH 8.0 </td><td> 1mM </td></tr></tbody></table> 表1:本协议中使用的缓冲区和介质的列表。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td> 上 </td><td> 关闭 </td></tr><tr><td>峰值功率</td><td> 150.0 </td><td> 2.5 </td></tr><tr><td>职责因素</td><td> 15.0 </td><td> 15.0 </td></tr><tr><td>循环/爆裂</td><td> 500 </td><td> 500 </td></tr><tr><td>循环次数</td><td colspan="2"> 28 </td></tr></tbody></table>表2:本协议中使用的剪切设置。这些条件已针对成熟的小鼠T细胞系进行了优化。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 抗体 </td><td> 供应商 </td><td> 抗体量/免疫沉淀 </td><td> 细胞数/免疫沉淀 </td><td> 洗涤条件 </td></tr><tr><td> H3 </td><td>阿布卡姆（ab1791） </td><td> 2.5毫克</td><td> 5 x 10 6 </td><td>一次在低盐缓冲液中，两次在高盐缓冲液中，两次在LiCl盐缓冲液中，三次在TE缓冲液中</td></tr><tr><td> H3K4me1 </td><td>阿布卡姆（ab8895） </td><td> 2.5毫克</td><td> 5 x 10 6 </td><td>一次在低盐缓冲液中，两次在高盐缓冲液中，两次在LiCl盐缓冲液中，三次在TE缓冲液中</td></tr><tr><td> H3K4me3的</td><td> Diagenode（pAb-003-050） </td><td> 2.5毫克</td><td> 5 x 10 6 </td><td>一次在低盐缓冲液中，两次在高盐缓冲液中，两次在LiCl盐缓冲液中，三次在TE缓冲液中</td></tr><tr><td> H3K27ac </td><td> Diagenode（pAb-174-050） </td><td> 2.5毫克</td><td> 5 x 10 6 </td><td> Oce在低盐缓冲液中，两次在高盐缓冲液中，两次在LiCl盐缓冲液中，三次在TE缓冲液中</td></tr><tr><td> IgG抗体</td><td> Diagenode（C15410206） </td><td>变量* </td><td>变量* </td><td>变量* </td></tr><tr><td colspan="5"> *在IgG对照的情况下，抗体和细胞以及洗涤步骤的量必须反映其他免疫沉淀的条件。 </td></tr></tbody></table> 表3:本研究中使用的抗体和洗涤条件。 <table border="1" fo:keep-together.within-page ="1"fo:keep-with-next.within-page ="always"&gt; <tbody><tr><td> 基因 </td><td> 正向引物 </td><td> 反向引物 </td><td> 探测 </td></tr><tr><td> Gapdh启动子 （ 0kb ） </td><td> 5&#39;-GGG TTC CTA TAA ATA CGG ACT GC-3&#39; </td><td> 5&#39;-CTG GCA CTG C​​AC AAG AAG A-3&#39; </td><td> 68 </td></tr><tr><td> Dtx1增强子 （+ 26kb ） </td><td> 5&#39;-CTC TGG GTT GTA GGG GAC AG-3&#39; </td><td> 5&#39;-GCA TGG GAA CTG TGT TAC AGA A-3&#39; </td><td> 27 </td></tr><tr><td> 基因沙漠 </td><td> 5&#39;-CAA TGC ATG GGT CCA GAT TT-3&#39; </td><td> 5&#39;-ATT GGC ACG GAA GTA GTG CT-3&#39; </td><td> 94 </td></tr></tbody></table> 表4:本研究中用于qPCR的引物和探针。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within页="总是"&gt; <tbody><tr><td> 问题 </td><td> 原因 </td><td> 解 </td></tr><tr><td rowspan="3">染色质处于剪切状态，碎片太大。 </td><td>已经使用了太多的细胞。 </td><td>减少细胞数量。 </td></tr><tr><td rowspan="2">剪切太低。 </td><td>增加剪切循环次数。 </td></tr><tr><td>增加剪切峰值功率，占空因数和/或周期/突发。 </td></tr><tr><td rowspan="3">染色质过度剪切，碎片太小。 </td><td>已经使用的细胞太少了。 </td><td>增加细胞数量。 </td></tr><tr><td rowspan="2">剪切太高了。 </td><td>减少剪切循环次数。 </td></tr><tr><td>降低剪切峰值功率，占空因数和/或周期/脉冲串。 </td></tr><tr><td rowspan="11">我的回收率太低了gG控制和/或阴性对照（高背景）。 </td><td>用于实验的染色质不足。 </td><td>增加染色质量。 </td></tr><tr><td>抗体量太低。 </td><td>增加抗体的量。 </td></tr><tr><td>抗体量过高。 </td><td>减少抗体的量。 </td></tr><tr><td>该抗体的特异性低。 </td><td>改变抗体。 </td></tr><tr><td rowspan="2">洗涤条件太严格。 </td><td>减少洗涤次数</td></tr><tr><td>降低洗涤缓冲液的严格性。 </td></tr><tr><td rowspan="2">洗涤条件不够严格。 </td><td>增加洗涤次数</td></tr><tr><td>增加洗涤缓冲液的严格性。 </td></tr><tr><td>在qPCR中吸取太多的DNA。 </td><td>减少qPCR中吸移的DNA量。 </td></tr><tr><td rowspan="2"> qPCR条件不是最优的。 </td><td>优化qPCR条件。 </td></tr><tr><td>减少循环次数。 </td></tr><tr><td rowspan="4">没有产品或产品在输入样品的qPCR反应中太低。 </td><td>在qPCR中没有足够的DNA被吸移。 </td><td>增加qPCR中DNA吸移量。 </td></tr><tr><td rowspan="2"> qPCR条件不是最优的。 </td><td>优化qPCR条件。 </td></tr><tr><td>增加循环次数。 </td></tr><tr><td>用于实验的染色质不足。 </td><td>增加染色质量。 </td></tr><tr><td rowspan="4">在阳性对照和/或panH3 ChIP中无信号。 </td><td>用于实验的染色质不足。 </td><td>增加染色质量。 </td></tr><tr><td>抗体量太低。 </td><td>增加抗体的量。 </td></tr><tr><td>反身体特异性低。 </td><td>改变抗体。 </td></tr><tr><td>洗脱是次优的。 </td><td>一定要频繁地旋转样品，以保持珠粒溶解。 </td></tr></tbody></table> 表5:故障排除。

Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines at JoVE.com

Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines

这项工作描述了使用成熟小鼠T细胞系的染色质免疫沉淀（ChIP）的方案。该方案适用于研究特定启动子位点或全基因组特异性组蛋白标记的分布。

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 ...

chromatin-immunoprecipitation-chip-in-mouse-t-cell-lines

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines

18.2K 次观看.  University of Giessen. 这项工作描述了使用成熟小鼠T细胞系的染色质免疫沉淀（ChIP）的方案。该方案适用于研究特定启动子位点或全基因组特异性组蛋白标记的分布。 

视频: Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines

Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a high content of structural proteins. Dissected limb muscles from adult mice are physically disrupted by mechanical homogenisation, or a combination of mincing and douncing, in a hypotonic buffer before formaldehyde fixation of the cell lysate. The fixed nuclei are purified by further cycles of mechanical homogenisation or douncing and sequential filtrations to remove cell debris. The purified nuclei can be sonicated immediately or at a later stage after freezing. The chromatin can be efficiently sonicated and is suitable for chromatin immunoprecipitation experiments, as illustrated by the profiles obtained for transcription factors, RNA polymerase II, and covalent histone modifications. The binding events detected using chromatin prepared by this protocol are predominantly those taking place in the muscle fiber nuclei despite the presence of chromatin from other fiber-associated satellite and endothelial cells. This protocol is therefore adapted to study gene regulation in the adult mouse skeletal muscle.

A novel protocol for the preparation of chromatin from adult mouse skeletal muscle adapted to the study of gene regulation in muscle fibers by chromatin immunoprecipitation is presented.

改进的小鼠骨骼肌染色质沉淀的协议

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

科研

生物化学

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 ...

遗传学

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

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 ...

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

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) is widely used to identify genome-wide binding locations of DNA-binding proteins. However, the ChIP-seq method is limited by its heterogeneity in length of sonicated DNA fragments and non-specific background DNA, resulting in low mapping resolution and uncertainty in DNA-binding sites. To overcome these limitations, the combination of ChIP with exonuclease digestion (ChIP-exo) utilizes 5&#8217; to 3&#8217; exonuclease digestion to trim the heterogeneously sized immunoprecipitated DNA to the protein-DNA crosslinking site. Exonuclease treatment also eliminates non-specific background DNA. The library-prepared and exonuclease-digested DNA can be sent for high-throughput sequencing. The ChIP-exo method allows for near base-pair mapping resolution with greater detection sensitivity and reduced background signal. An optimized ChIP-exo protocol for mammalian cells and next-generation sequencing is described below.

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

Precise determination of protein-binding locations across the genome is important for understanding gene regulation. Here we describe a&#160;genomic mapping method that treats chromatin-immunoprecipitated DNA with exonuclease digestion (ChIP-exo) followed by high-throughput sequencing. This method detects protein-DNA interactions with near base-pair mapping resolution and high signal-to-noise ratio in mammalian neurons.

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

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

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

在激活人类细胞中的基因表达的动态检测组蛋白修饰染色质免疫沉淀（ChIP）

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

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 ...

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 ...

Chromatin Extraction from Frozen Chimeric Liver Tissue for Chromatin Immunoprecipitation Analysis

Crosslinking Chromatin Immunoprecipitation (X-ChIP) is a widely used technique to assess levels of histone marks and occupancy of transcription factors on host and/or pathogen chromatin. Chromatin preparation from tissues creates additional challenges that need to be overcome to obtain reproducible and reliable protocols comparable to those used for cell culture. Tissue disruption and fixation are critical steps to achieve efficient shearing of chromatin. Coexistence of different cell types and clusters may also require different shearing times to reach optimal fragment size and hinders shearing reproducibility. The purpose of this method is to achieve reliable and reproducible host chromatin preparations from frozen tissue (liver) suitable for both ChIP-qPCR and next generation sequencing (NGS) applications. We observed that the combination of liquid nitrogen tissue pulverization followed by homogenization leads to increased reproducibility compared to homogenization only, since it provides a suspension consisting mostly of dissociated single cells that can be efficiently sheared. Moreover, the fixation step should be performed under mild rotation to provide homogeneous crosslinking. The fixed material is then suitable for buffer-based nuclei isolation, to reduce contamination of cytoplasmic protein and pathogen DNAs and RNAs (when applicable), avoiding time-consuming centrifugation gradients. Subsequent sonication will complete nuclear lysis and shear the chromatin, producing a specific size range according to the chosen shearing conditions. The size range should fall between 100 and 300 nt for NGS applications, while it could be higher (300-700 nt) for ChIP-qPCR analysis. Such protocol adaptations can greatly improve chromatin analyses from frozen tissue specimens.

This protocol focuses on chromatin preparation from snap frozen tissues and it is suitable for Crosslinking Chromatin Immunoprecipitation (X-ChIP) followed by either quantitative PCR analysis (X-ChIP-qPCR) or next generation sequencing approaches (X-ChIP-seq).

从冷冻嵌合肝组织中提取染色质，用于染色质免疫沉淀分析

Crosslinking Chromatin Immunoprecipitation (X-ChIP) is a widely used technique to assess levels of histone marks and occupancy of transcription factors on ...