我们感谢Karyn Esser，Nobuya小池和Noheon Park提供有用的建议。这项工作部分由NIH / NIGMS（R01GM114424）向S.-HY和Robert A.Welch基金会（AU-1731）和NIH / NIA（R01AG045828）向ZC提供支持

动物护理在机构动物护理和使用委员会（IACUC）指南下进行，并且程序是根据得克萨斯州休斯敦大学健康科学中心批准的动物方案进行的。 从交联骨骼肌中分离核<ol><li>称重和切碎后肢骨骼肌，从大约20周龄的C57BL / 6雄性小鼠分离，在冰冷的磷酸盐缓冲盐水（PBS）中，如先前详细描述的22所述 。 注意:我们通常从一只小鼠获得1.0 - 1.5 g骨骼肌。 <ol><li>将切碎的骨骼肌组织放入含有10mL PBS的50mL锥形管中。 </li></ol></li><li>在4℃下以300xg离心5分钟。 </li><li>通过抽吸小心清除PBS上清液。 </li><li>通过使用具有1,2,3和4mL水的参考管估计每个50mL管中的颗粒体积。 </li><li>添加7卷冰冷的1％甲醛在PBS中，并使用探针组织匀浆器在冰上匀化样品（ 参见表材料 ）。 注意:可选:均质程序可在冷藏室进行。 </li><li>通过在室温下孵育10分钟将样品交联。 </li><li>通过加入1M甘氨酸使终浓度为0.125M的交联反应骤冷，并在室温下孵育5分钟。 </li><li>在4℃下将样品以3,000xg离心5分钟。通过抽吸去除上清液。 </li><li>用含有新鲜蛋白酶抑制剂（0.2mM苯甲基磺酰氟（PMSF））的10mL冰冷碱性缓冲液（pH7.3的10mM HEPES-KOH，10mM EDTA，10mM KCl，5mM MgCl 2，0.5mM DTT） ，和10μg/ mL亮抑酶肽）。在4℃下以3,000g离心5分钟，并通过抽吸小心地去除上清液。 </li><li>将沉淀重悬于6mL裂解缓冲液（10mM HEPES-KOH，含有新鲜蛋白酶抑制剂（0.2mM PMSF和10μg/ mL亮抑酶肽）的H 7.3，10mM EDTA，10mM KCl，5mM MgCl 2，0.5mM DTT，1％Triton X-100）。 </li><li>将样品转移到预先制备的15 mL Dounce匀浆器中，并在冰上孵育10分钟。在冰上均匀化每个样品，使用松散的杵进行15次缓慢的冲程，然后用紧密的杵将15次冲击以释放细胞核。 </li><li>通过细胞过滤器（孔径:100μm）过滤匀浆（6 mL），并用4 mL裂解缓冲液冲洗。在4℃下将样品以1,000xg离心10分钟。重悬于5 mL冰冷的基础缓冲液中，用紧密的杵将其重复10次以释放细胞核。 <ol><li>用分光光度计（OD260 / OD280）取出20μL的DNA浓度测定等分试样，必要时用台盼蓝进行显微镜观察（见下文）（ 图 1A ）。 </li></ol></li><li>过滤悬浮液细胞过滤器（孔径:70μm），如上所述用2mL碱缓冲液再次冲洗管并过滤。 </li><li>重复过滤步骤1.13，细孔过滤器逐渐减小孔径（40μm，30μm，20μm和10μm）。 注意:在步骤1.12-1.14中总共使用6孔径的过滤器。 </li><li>在4℃下以1,000g离心10分钟。去除上清液，并将沉淀物重悬于500μL碱性缓冲液中。 </li><li>测量如上所述的DNA浓度。 注意:从一只动物的两只后肢组合获得约200μg的核酸DNA。任选地，用台盼蓝染色（库存浓度0.4％，1:5稀释）显微镜观察保存20μL;核会染蓝色（ 图1B ）。 </li><li>在4℃下以1,000g离心10分钟，弃上清。将沉淀物储存在-80°C。 </li></ol>超声处理 &lt;OL&gt; <li>在500μLSDS裂解缓冲液中解冻样品，并轻轻移液重悬。 </li><li>将核DNA悬浮液转移到冰上的玻璃小瓶中。 </li><li>从碟形传感器运行超音波超声波声能。有关设备详细信息，请参见材料表 。使用以下设置:每个突发周期:200;强度:5;占空比:20;温度4-6℃; 30秒开/关。 注:评估超声功效的实例如图2所示。 </li><li>将超声处理的染色质转移到1.5 mL管中。以12,000 xg离心15分钟，并将上清液转移到新管中。将50μL超声处理的样品（〜7 - 10μg/肌肉样品）转移到新的1.5 mL管中，以在65°C进行反向交联过夜。将剩余的样品在-80℃下冷冻。 </li></ol>超声和定量评估<ol><li>添加1.0μL的RNA酶A（500 U / mL RNA酶A:20,000 U / mL RNase T1）保存在50μL超声波样品中，37℃孵育30 min。 </li><li>加入8μL蛋白酶K（10mg / mL），并在55℃下孵育30分钟。 </li><li>通过DNA提取试剂盒收获DNA。 <ol><li>加入5卷结合缓冲液，应用于旋转柱，离心4,000 xg，10分钟。 </li><li>将通过应用到同一列并再次离心。 </li><li>用洗涤缓冲液洗涤两次，每次13,000 xg，1分钟。 </li><li>离心再次以13,000 xg，1分钟干燥柱。 </li><li>加入50μLH 2 O，离心13000 x g，1 min洗脱DNA。 </li><li>将通过应用到同一列并再次离心。 </li></ol></li><li>用分光光度计（OD260 / OD280）定量DNA量。在0.8％凝胶上运行样品以评估超声产品的大小和量（1μg）（ 图2 ）。 注意:平均染色质产量约为〜20％。 </li></ol> ChIP <ol><li>将以前保存的染色质在-80℃下解冻，并将染色质等分至〜100〜120μg/管。使用放射免疫沉淀测定（RIPA）缓冲液（20mM Tris-HCl（pH 7.4），150mM NaCl，1mM EDTA，1mM NaF，1mM Na 3 VO 4，1mM PMSF，1mM蛋白酶抑制剂混合物（PIC），0.1％脱氧胆酸盐w / v），1％Triton X-100）。 <ol><li>如果每组中有多个样品，则将等量的染色质DNA组合至最终量为100 - 120μg/样品。节省10％的输入。 </li></ol></li><li>加入BSA-pre-blocked（〜2 h，4℃）蛋白A / G琼脂糖（非鸡抗体）或IgY珠（鸡抗体）的40μL（RIPA缓冲液中的50％v / v浆液），并孵育在4℃下旋转3小时。 </li><li>以1,000 xg，10分钟离心，小心地将上清液转移到新管中。 </li><li>每1μg抗体加1μg抗体25-100μg染色质DNA。 </li><li>以约15rpm在4℃下轻轻旋转过夜。 </li><li>加入10μLBSA-预封闭（〜2 h，4°C）蛋白A / G琼脂糖（非鸡抗体）或IgY珠（鸡抗体）混合，并在冷藏室中旋转2 h。 </li><li>洗珠如下。用1mL RIPA缓冲液洗涤3分钟两次，用1mL高盐缓冲液（20mM Tris-HCl（pH7.4），500mM NaCl，2mM EDTA，1mM PMSF，1％Triton X-100）洗涤3分钟两次，用1mL LiCl缓冲液（20mM Tris-HCl（pH7.4），250mM LiCl，2mM EDTA，1mM PMSF，1％脱氧胆酸钠，0.5％NP40）洗涤3分钟两次。 <ol><li>用1mL Tris-EDTA（TE）缓冲液（10mM Tris-HCl（pH7.4），1mM EDTA）洗涤3分钟一次。然后用洗脱缓冲液（20mM Tris-HCl，5mM EDTA和0.5％SDS）洗脱DNA。 </li></ol></li><li>以1,000 g离心1分钟，小心取出上清液。 </li><li>用50μL洗脱缓冲液重悬珠。 </li><li>孵化10min，65℃。 </li><li>以12,000克离心5分钟。 </li><li>将上清液转移到新管中，再加入50μL，再次以12,000 g离心5分钟。最终洗脱体积为100μL。 </li><li>反向交联和DNA洗脱。 <ol><li>将洗脱的染色质在65℃孵育过夜。 </li><li>加入1.0μLRNA酶A，37℃孵育30 min。 </li><li>加入8μL蛋白酶K（10mg / mL），并在55℃下孵育30分钟。 </li><li>通过使用PCR清洗试剂盒，根据制造商的方案，以50μL的洗脱体积收获DNA。 </li></ol></li><li>通过实时qPCR分析配置文件，如前所述25,26 。 注意:引物序列如图3所示 。数据以平均值±SEM表示（ 图3 ）。 </li></ol>

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作者没有什么可以披露的。

在这里，我们描述了一种使用交联骨骼肌组织分离高质量细胞核的可靠方法。进行顺序过滤以有效地将细胞核与碎片分离，来自碟形换能器的超声波能量剪切染色质进行ChIP分析。结果显示BMAL1与靶启动子的昼夜节律时间特异性结合。 当交联发生时，ChIP可用于捕获基因组DNA上的实时蛋白质占用。为了利用这种潜力，我们旨在开发一种方法，允许在组织解剖时交叉骨骼肌，并简化细胞核分离而不进行梯度超速离心。由于与软组织如肝脏相比，纤维丰富的骨骼肌的均质化困难，我们在冰冷的PBS中切碎肌肉组织，然后将样品在甲醛缓冲液中匀化。淬火后，组织悬浮液离心并用冰冷的碱缓冲液冲洗，以清除任何剩余的甲醛。通过Dounce匀浆释放细胞核，依次过滤匀浆，逐渐去除细胞碎片和肌原纤维。我们设计了一系列过滤以最大限度地减少过滤器堵塞，这可能不利地影响产量。当顺序过滤完成时，只剩下非常短的肌原纤维。 超声处理和ChIP程序从以前的报告12中进行了改进，包括超声处理时间和SDS缓冲液量。碟形超声波清洗机允许在冷水浴中将玻璃小瓶中的样品暴露于集中式超声波。与探头超声波探头相比，该超声波发生器控制样品温度以避免过热，也可防止样品交叉污染。如果使用探针超声波仪器，则需要根据经验确定最佳超声波条件。我们也减少了t的SDS缓冲液，因为肌肉染色质的产量低于肝脏12 。几种方案28,31,32包括在超声处理之前在冰上或在室温下孵育。然而，根据我们的经验，在冰上预孵育没有提高超声处理效率。事实上，在某些情况下，超声处理受到损害。残留的肌原纤维可能在培养期间纠缠染色质DNA并减弱超声功能。在SDS裂解缓冲液中核悬浮后立即进行超声处理，我们成功地获得渐进的染色质片段化，增加超声波循环（ 图2 ）。 我们用ChIP qPCR验证了染色质的质量。 如图3所示，BMAL1启动子占有率在ZT6时是鲁棒的，在ZT18处是最小的，与先前显示的BMAL1大约一致dian启动子结合18 。该功能测定证实了核和染色质的质量。近年来，NGS的快速发展为ChIP应用开辟了新的视野，ChIP-seq可以高灵敏度定量询问基因组结合17 。特别是对于ChIP-seq，需要高质量的细胞核和染色质来一致地捕获蛋白质 - DNA相互作用。本文描述的程序可能构成使用骨骼肌的ChIP-seq研究的宝贵资源。值得注意的是，ChIP-seq库准备需要额外的措施来提高信号分辨率，如基于PAGE的尺寸选择18 。 总之，我们开发了一种基于过滤的方案，从交联骨骼肌制备高质量的细胞核。我们消除了超速离心的需要，使其易于应用。除了感兴趣的基因的ChIP，核和染色质如上所述，可以广泛适用于ChIP-seq研究。

骨骼肌在生理和行为中起重要作用。多核肌纤维由肌原纤维组成，其中肌动蛋白和肌球蛋白形成称为sarcomeres的功能单位以产生收缩力。骨骼肌也是身体中最大的代谢器官，占餐后葡萄糖摄入量的80％以上，调节胰岛素反应和代谢体内平衡1,2 。肌肉生理和代谢受到昼夜节律钟的限制，内在的生物计时器3,4,5,6。例如， Bmal1的骨骼肌特异性缺失，核心昼夜节律钟成分之一，导致骨骼肌中胰岛素抵抗和葡萄糖摄取减少，发现动物发生2型糖尿病7 。一世此外，骨骼肌也越来越被认为是内分泌器官8 ，分泌肌动蛋白以调节全身代谢和生理学。机械研究需要完全了解骨骼肌中的这些调节功能。 ChIP是描述DNA结合蛋白启动子募集的有力方法。最初开发了ChIP以鉴定染色质DNA 9,10上的核小体组织。据报道，使用甲醛，硫酸二甲酯或紫外线照射（UV） 11,12来交联蛋白质和染色质DNA的各种方法。甲醛交联是最常用的，保存染色质结构和DNA-蛋白相互作用9,13,14 。交联色谱通过超声处理切片并用针对感兴趣的特定DNA结合蛋白15,16的抗体进行免疫沉淀。近年来，已经开发了ChIP-sequencing（ChIP-seq），一种将ChIP与NGS相结合的方法，用于询问全基因组转录因子结合17 ，并且在某些情况下监测时间过程18,19,20的动态变化。例如，昼夜节律ChIP-seq研究已经显示出昼夜节律时钟组分和组蛋白标记物的基因组结合的高度编排的序列，其在整个〜24小时昼夜循环中驱动时间上精确的基因表达18 。 大多数可用的ChIP协议设计用于软组织（ 例如肝，脑等 ），并且很少已经出版用于硬组织，包括ke伸肌在富含纤维的骨骼肌中均匀化并分离高质量细胞核21是技术上的挑战，特别是对于需要交联的ChIP实验。在最近的肌肉ChIP研究22中 ，卫星细胞与肌纤维分离，并且通过涉及组织消化的延长的过程从两种细胞类型制备细胞核。在分离的核上进行甲醛交联之前，整个过程需要大约三个小时才能完成。虽然这种方法避免了交联肌纤维，这使得肌肉组织更有效地进行高效均匀化，并且能够产生高质量的细胞核，但是从组织收集到细胞核交联的显着的时间滞后引起了DNA变化的风险蛋白质相互作用。相比之下，大多数研究在实验治疗或组织收集后立即进行交联，以保存实时DNA-蛋白质n约束12 。在交联前核分离的第二个缺点是它排除了时间敏感的应用，例如通常以3-4小时间隔发生的昼夜样本采集。没有交联核，分离需要在解剖后立即进行，而交联样品可以在整个时间过程完成后一起处理，从而确保更大的实验一致性。 还报道了与未交联的骨骼肌分离核的其它方案。两项研究描述了使用梯度超速离心将细胞核与剩余的肌原纤维和细胞碎片分开23,24 。虽然蔗糖或胶体梯度超速离心对未交联的肌肉组织有效，但我们的实验表明，交联后，梯度超速离心不能将细胞从细胞分离出来ebris在梯度上。 因此，我们开发了一种使用交联的小鼠骨骼肌组织分离高质量核的方法。而不是梯度超速离心，我们设计了一种串联过滤方法来有效地将细胞核与碎片分离。经超声处理后，染色质样品成功应用于ChIP研究，显示BMAL1蛋白与靶启动子结合的昼夜节律模式。我们的方法可以广泛适用于肌肉组织的各种机械研究。

<table><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Formaldehyde solution</td><td>Sigma Aldrich</td><td>F8775&amp;nbsp;</td><td></td></tr><tr><td>Glycine</td><td>Fisher Scientific</td><td>BP381-5</td><td></td></tr><tr><td>Trypan Blue solution (0.4%)</td><td>Fisher Scientific</td><td>15250061</td><td></td></tr><tr><td>RNase A</td><td>Sigma Aldrich</td><td>10109142001</td><td></td></tr><tr><td>Protease K</td><td>Sigma Aldrich</td><td>3115887001</td><td></td></tr><tr><td>Chicken Anti-BMAL1 antibody</td><td></td><td></td><td>Generated in chicken (Cocalico Biologicals) against antigen aa 318 - 579, and IgY was affinity purified using the same antigen.&amp;nbsp;</td></tr><tr><td>Chicken IgY Precipitating Resin</td><td>GenScript</td><td>L00405</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>KINEMATICA&amp;nbsp;Polytron PT2100 Benchtop Homogenizer</td><td>Fisher Scientific</td><td>08-451-178</td><td></td></tr><tr><td>15 mL Dounce tissue grinder</td><td>Whearton</td><td>357544</td><td></td></tr><tr><td>Falcon Cell Strainers 100 &amp;micro;m</td><td>Fisher Scientific</td><td>08-771-19</td><td></td></tr><tr><td>Falcon Cell Strainers 70 &amp;micro;m</td><td>Fisher Scientific</td><td>08-771-1</td><td></td></tr><tr><td>Falcon Cell Strainers 40 &amp;micro;m</td><td>Fisher Scientific</td><td>08-771-2</td><td></td></tr><tr><td>pluriStrainer 30 &amp;micro;m</td><td>PluriSelect</td><td>43-50030-03</td><td></td></tr><tr><td>pluriStrainer 20 &amp;micro;m</td><td>PluriSelect</td><td>43-50020-03</td><td></td></tr><tr><td>pluriStrainer 10 &amp;micro;m</td><td>PluriSelect</td><td>43-50010-03</td><td></td></tr><tr><td>Covaris S2 Focused-ultrasonicator</td><td>Covaris</td><td>Model S2</td><td></td></tr><tr><td>Labquake&amp;nbsp;</td><td>Thermo Scientific</td><td>C415110</td><td></td></tr><tr><td>CCD Microscope Camera</td><td>&amp;nbsp;Leica Microsystems</td><td>DFC3000 G</td><td></td></tr><tr><td>&lt;strong&gt;Reagent Kit&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DNA extraction kit</td><td>Thermo Scientific</td><td>K0691</td><td></td></tr><tr><td>&lt;strong&gt;Buffers&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>All buffer components are discribed in the protocol. Each component was purchased from Sigma Aldrich</td><td></td><td></td><td></td></tr></tbody></table>

a filtration-based method of preparing high-quality nuclei from cross-linked skeletal muscle for chromatin immunoprecipitation

染色质免疫沉淀（ChIP）是确定蛋白质结合染色质DNA的有效方法。然而，富含纤维的骨骼肌由于技术上难以隔离高质量的细胞核而且对肌原纤维的污染最小，因此一直是ChIP的挑战。以前的方案尝试在交联之前纯化细胞核，这导致在延长的细胞核制备过程中DNA-蛋白质相互作用发生改变的风险。在目前的方案中，我们首先交联从小鼠收集的骨骼肌组织，并将组织切碎并超声处理。由于我们发现超速离心不能使用交联肌肉组织将原核与肌原纤维分开，我们设计了一个顺序的过滤程序，以获得没有显着肌原纤维污染的高质量细胞核。我们随后使用超声波仪制备了染色质，并且使用抗BMAL1抗体的ChIP测定显示强大的昼夜节律结合目的基因启动子的BMAL1模式。该过滤方案构成了将高质量细胞核与交联骨骼肌组织隔离的一种易于应用的方法，允许对昼夜节律和其他时间敏感性研究进行一致的样品处理。结合下一代测序（NGS），我们的方法可以部署用于骨骼肌功能的各种机械和基因组研究。

这里我们在组织收集后立即进行甲醛交联，以保持实时的DNA-蛋白质相互作用。然而，我们发现通常用于细胞核分离的蔗糖或胶体梯度23,24在分离细胞核和肌原纤维方面无效（数据未显示）。原因可能是交联赋予核和肌原纤维相似的重力。因此，我们开发了一个串联过滤方法，以有效地从核部分去除大的肌原纤维和其他碎屑。经过100μm过滤后，仍有较大的组织碎屑和肌原纤维（ 图 1A ）。相比之下，在顺序过滤结束时，大部分大的组织碎片，完整的细胞和大的肌原纤维被成功地去除（ 图1B ）。 27或小鼠胚胎成纤维细胞（MEF） 28的超声处理，但在我们的经验中，它干扰了骨骼肌细胞核的超声处理，并导致广泛的涂片无效超声处理使用目前的方案，在SDS裂解缓冲液中悬浮后立即进行超声处理，我们观察到有效的超声处理与染色质以循环依赖的方式逐渐切碎，最终产生〜500bp的DNA片段（ 图2 ）。裂解缓冲液中的预孵育可以显着地破坏超声处理效率。 诈骗确定ChIP染色质制备的质量，我们检测了昼夜节律转录因子BMAL1的DNA结合，其分别在Zeitgeber时间（ZT）6和ZT18处显示结合峰和谷18,29。在ZT6和ZT18处收集C57B / 6J小鼠的骨骼肌样品，如上制备染色质样品。简言之，超声处理后，用BSA预先封闭的IgY珠粒预先清除切碎的染色质样品，然后在4℃下与抗BMAL1抗体孵育过夜。洗脱和纯化染色质后，我们进行RT-qPCR检测两个目标基因Nr1d1和Dbp 30的 E-Box元件上的BMAL1结合。我们检测到BMAL1与ZT6上的E盒元件的稳定结合，ZT18处的最小结合（ Nr1d1 :0.452±0.022与0.039±0.002， Dbp -0.4:0.627±0.013对比0.062±0.009， Dbp+0.8:0.176±0.013对0.008±0.001， Dbp +2.4:0.466±0.010对0.122±0.014;所有值均为±SEM）（ 图3 ），验证了骨骼肌中时间敏感转录因子结合分析方案。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/56013/56013fig1.jpg" /> 图1 :顺序过滤有效去除组织碎片。 （A）在100μm过滤后显示样品的代表性图像。观察到大的组织和纤维碎片。 （B）连续过滤后显示样品的代表性图像。大纤维碎片被清除。仅观察到孤立的核和小肌原纤维碎片。使用10X，20X和40X放大倍数的光学显微镜拍摄照片。比例尺显示在右侧面板上。<a href="http://ecsource.jove.com/files/ftp_upload/56013/56013fig1large.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/56013/56013fig2.jpg" /> 图2 :通过10个循环超声处理逐步染色质粉碎。如在0.8％琼脂糖凝胶中显示的，将消化的染色质DNA超声处理至约500bp的10个循环，在150V下运行60分钟。右面板表示在冰冷的SDS裂解缓冲液中预孵育1小时后的较低的超声处理效率。 <a href="http://ecsource.jove.com/files/ftp_upload/56013/56013fig2large.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/56013/56013fig3.jpg" /> 网络连接第3组:在ZT6和ZT18处收集的具有小鼠骨骼肌样品的BMAL1 ChIP的代表性qPCR结果。数据以平均值±SEM表示。 Dbp -0.4，+0.8和+2.4表示Dbp基因上E-Box元件的位置。 NC:IgY阴性对照。 BMAL1结合的时间模式与先前在ZT618周围显示BMAL1结合峰的结果一致。正向和反向引物如下。 Rev-erba:5&#39;-GTAGACTACAAATCCCAACAATCCTG和5&#39;-TGGAGCAGGTACCATGTGATTC; Dbp -0.4:5&#39;-ACACCCGCATCCGATAGC和5&#39;-CCACTTCGGGCCAATGAG; Dbp +0.8:5&#39;-ATGCTCACACGGTGCAGACA和5&#39;- CTGCTCAGGCACATTCCTCAT; Dbp +2.4:5&#39;-TGGGACGCCTGGGTACAC和5&#39;-GGGAATGTGCAGCACTGGTT。 <a href="http://ecsource.jove.com/files/ftp_upload/56013/56013fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation at JoVE.com

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

我们提出了基于过滤的方案，以将高质量的细胞核与交联的小鼠骨骼肌分离，其中我们消除了超离心的需要，使其易于应用。我们显示从核制备的染色质适用于染色质免疫沉淀和可能的染色质免疫沉淀测序研究。

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 ...

a-filtration-based-method-preparing-high-quality-nuclei-from-cross

Nanyang Technological University

Research

JoVE Journal

Biochemistry

8.8K 次观看.  University of Texas Health Science Center at Houston. 我们提出了基于过滤的方案，以将高质量的细胞核与交联的小鼠骨骼肌分离，其中我们消除了超离心的需要，使其易于应用。我们显示从核制备的染色质适用于染色质免疫沉淀和可能的染色质免疫沉淀测序研究。 

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

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

Quantitative Analysis of Chromatin Proteomes in Disease

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due to the high percentage of binucleated cells,1 the predominantly heterochromatic state of the DNA, and the non-dividing nature of the cardiomyocyte which renders adult nuclei in a permanent state of interphase.2 Transcriptional regulation during development and disease have been well studied in this organ,3-5 but what remains relatively unexplored is the role played by the nuclear proteins responsible for DNA packaging and expression, and how these proteins control changes in transcriptional programs that occur during disease.6 In the developed world, heart disease is the number one cause of mortality for both men and women.7 Insight on how nuclear proteins cooperate to regulate the progression of this disease is critical for advancing the current treatment options.
Mass spectrometry is the ideal tool for addressing these questions as it allows for an unbiased annotation of the nuclear proteome and relative quantification for how the abundance of these proteins changes with disease. While there have been several proteomic studies for mammalian nuclear protein complexes,8-13 until recently14 there has been only one study examining the cardiac nuclear proteome, and it considered the entire nucleus, rather than exploring the proteome at the level of nuclear sub compartments.15 In large part, this shortage of work is due to the difficulty of isolating cardiac nuclei. Cardiac nuclei occur within a rigid and dense actin-myosin apparatus to which they are connected via multiple extensions from the endoplasmic reticulum, to the extent that myocyte contraction alters their overall shape.16 Additionally, cardiomyocytes are 40% mitochondria by volume17 which necessitates enrichment of the nucleus apart from the other organelles. Here we describe a protocol for cardiac nuclear enrichment and further fractionation into biologically-relevant compartments. Furthermore, we detail methods for label-free quantitative mass spectrometric dissection of these fractions-techniques amenable to in vivo experimentation in various animal models and organ systems where metabolic labeling is not feasible.

Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

在疾病的染色质蛋白质组的定量分析

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due ...

医学

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 ...

生物学

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

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

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

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

代高通量测序技术（ChIP-seq的高品质的染色质免疫沉淀DNA模板）

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

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

该ChroP方法结合芯片和质谱剖析染色质的基因座特异性蛋白质组学风景

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

产生和人类INO80染色质重塑复合物及其亚纯化

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.

MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

使用串联亲和纯化的MyoD相互作用组鉴定质谱分析法

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ...

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

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

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

该芯片外型方法:确定蛋白质-DNA相互作用与近基部精密配对

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

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 ...

用于 Multiome 10X 基因组学分析的高质量样品制备

High-Quality Brain and Bone Marrow Nuclei Preparation for Single Nuclei Multiome Assays

Single-cell analysis has become the approach of choice for unraveling the complexity of biological processes that require assessing the variability of individual cellular responses to treatment or infection with single-cell resolution. 
Many techniques for single-cell molecular profiling have been developed over the past 10 years, and several dedicated technologies have been commercialized. The 10X Genomics droplet-based single-cell profiling is a widespread technology that offers ready-to-use reagents for transcriptomic and multi-omic single-cell profiling. The technology includes workflows for single-cell and single-nuclei RNA sequencing (scRNA-Seq and snRNA-Seq, respectively), scATAC-Seq, single-cell immune profiling (BCR/TCR sequencing), and multiome. The latter combines transcriptional (scRNA-Seq) and epigenetic information (scATAC-Seq) coming from the same cell. 
The quality (viability, integrity, purity) of single-cell or single-nuclei suspensions isolated from tissues and analyzed by any of these approaches is critical for generating high-quality data. Therefore, the sample preparation protocols should be adapted to the particularities of each biological tissue and ensure the generation of high-quality cell and nuclei suspensions.
This article describes two protocols for preparing brain and bone marrow samples for the downstream multiome 10X Genomics pipeline. The protocols are performed stepwise and cover tissue dissociation, cell sorting, nuclei isolation, and quality control of prepared nuclei suspension that is used as starting material for cell partitioning and barcoding, library preparation, and sequencing. These standardized protocols produce high-quality nuclei libraries and robust and reliable data.

作者聚焦：通过单细胞分析推进生物医学研究 

The success of single-cell/single-nuclei transcriptomics and multi-omics largely depends on the quality of cells/nuclei. Therefore, isolating cells/nuclei from tissue and their purification must be highly standardized. This protocol describes the preparation of nuclei from the brain and bone marrow for downstream single-nuclei multiome assay.

用于单核多组学检测的高质量脑和骨髓核制备

Single-cell analysis has become the approach of choice for unraveling the complexity of biological processes that require assessing the variability of ...