We thank the Venters Lab and members of the Molecular Physiology and Biophysics Department for helpful discussions. Special thanks to Frank Pugh for his guidance, mentoring, and many insightful discussions on the nuances of ChIP-exo while I was a post-doctoral fellow in his laboratory.

注:双蒸H 2 O或等值级分子被推荐用于所有的缓冲区和反应混合物。 0天:材料制备和电池丰收 1.缓冲液配制<ol><li>准备裂解缓冲液1 - 3（ 表1 - 3），并添加100微升完整的蛋白酶抑制剂股票（CPI）为各50毫升缓冲液在使用前的。通过溶解一片1毫升分子级的H 2 O.准备CPI股票</li><li>准备内部缓冲器（ 表4 - 7）。加入100微升CPI股票至50ml阻塞和RIPA缓冲液。不要添加到CPI的Tris和芯片洗脱缓冲液。 </li></ol> 2.适配器寡核苷酸退火注:特定的寡核苷酸序列可以在附加信息部分找到。 <ol><li>准备适配器退火混合物（ 表8 - 9年 ）。涡旋混合并短暂旋管收集的内容。分装100微升每个混合成0.5毫升管。 </li><li>通过运行在热循环仪程序（ 表10）杂交的寡核苷酸。 商店在-80℃下退火的寡核苷酸。 </li></ol> 3. 在体内染色质交联与甲醛注:对于典型的ChIP实验，原料应包含约50万个细胞。 <ol><li>加入新鲜37％不含甲醇的甲醛原液磷酸盐缓冲盐水（PBS）洗涤细胞培养物的1％（V / V），调匀，并让在室温下放置10分钟的最终浓度。 注意:例如，我们通常在50毫升室温的PBS加1.4毫升37％不含甲醇的甲醛至细胞。此外，避免甲醛交联传媒自媒体的蛋白质会熄灭一些甲醛。 </li><li>淬火加2.5M的甘氨酸交联反应E对于125毫米的终浓度。调匀。 </li><li>细胞转移到50毫升管冰。旋转细胞5分钟以1000 xg离心在4℃下。倒出上清。 </li><li>通过移液加入1毫升冰冷的1×PBS中至细胞沉淀重悬。转移到1.5mL管中。 </li><li>自旋细胞3分钟以2000 xg离心在4℃下。吸上清。 </li><li>马上闪1.5毫升管用液态氮冷冻细胞沉淀。存储在-80℃。交联细胞是无限稳定在-80℃。 </li></ol>第1天:细胞裂解，超声处理以及芯片 4.细胞裂解注:在所有的细胞裂解段，样品必须保持在冰上或4℃，以减少交联逆转。 <ol><li>简言之解冻交联细胞沉淀。彻底重悬每个粒料在0.5ml裂解缓冲液1和用4.5ml裂解缓冲液1在15ml聚苯乙烯管结合。 </li><li>岩管10分钟，4℃为在摇摆平台上。旋在2000 xg离心在4℃下4分钟。倒出上清。 </li><li>彻底悬浮颗粒每0.5ml的裂解液2，并添加4.5毫升裂解液2次重悬。 </li><li>在4℃下摇动5分钟在摇摆平台上。旋在2000 xg离心在4℃下5分钟。倒出上清，轻轻拍打过量对纸巾。 </li><li>彻底悬浮颗粒每0.5ml的裂解液3和加入1 ml裂解液3次重悬。保持冰核裂解液并立即进行超声波处理。 </li></ol> 5.超声核裂解液注:在所有的超声波段，样品必须保持在冰上，以尽量减少交联逆转。在与下面的协议结合使用超声波仪的具体型号可能会在材料表中找到。特定的声波仪的使用和其他仪器的指导方针的细节可以在附加信息部分找到。 <ol><li> PL含核提取物15毫升聚苯乙烯管王牌共振适配器（金属条不应该接触管壁）。 </li><li>超声处理核裂解液在冰冷水浴2×15分钟的会议以30秒开/ 30秒关在中等功率。在一个时间只有超声处理两个15毫升管。这些设置在广泛的细胞系和原代细胞类型工作，但如果染色质剪切是不完全的，可能需要进一步优化。 </li><li>要检查超声结果，转让2×10微升样本裂解成1.5毫升管。 <ol><li>扭转交联，用10微升TE-的RNaseA并在37℃下0.2微升蛋白酶K.孵育的30分钟结合第一10微升等分试样。加入4微升6X二甲苯DNA染料。 </li><li>为了保持交联，与2微升6X二甲苯DNA染料结合第二个10微升等分。 </li><li>在140 V代表约30运行上的1.5％琼脂糖凝胶两个样品 - 45分钟，直到在梯形溴酚染料我的一路下跌凝胶小号¾。 </li></ol></li><li>如果超声成功（剪切至100个最DNA片段 - 500基点），转让超声裂解液含有150微升10％的Triton X-100的2ml管。旋涡混合。 </li><li>以沉淀不溶染色质和碎屑，旋超声核裂解物10分钟，以20,000 xg离心在4℃下。 </li><li>转移上清液至新的2ml管中。在-80℃，立即着手芯片或存储样本下去。超声处理提取物不应该被冷冻和解冻不止一次。 </li></ol> 6.耦合抗体珠注意:不要旋涡或冻结/解冻磁珠因为他们将打破，并增加背景信号。 <ol><li>充分混合股票，分装ÿ微升珠浆到1.5mL管中后，其中Y = 1.1×2.5微升X（多家芯片样品）。 2.5微升珠浆结合能力高达13微克的IgG。洗合并的珠用1ml阻断3倍缓冲。 </li><li>对于洗涤后的珠更一致aliquotting，在原来的10倍浆液的体积（25微升/芯片）用封闭缓冲液重悬珠。分装25微升每芯片的样品放入1.5毫升管。 </li><li>加入5 - 10微克抗体相应的重悬珠等分。最终带来量高达250微升用封闭缓冲。在4℃孵育的样品4小时（或者，过夜16小时）的旋转平台上。 </li><li>吸上清。为了除去未结合的或过剩的抗体，用1毫升封闭缓冲液洗珠一次。 </li><li>吸最后一次洗涤后，立即加入超声提取物（〜1.6ml）中的5000万细胞当量抗体:珠缀合物。如果超声提取物是没有准备好，在100微升封闭缓冲液重悬珠，直到他们准备好了。关键是要永远让珠干燥。 </li></ol> 7.染色质免疫沉淀（ChIP） <ol><li>每个芯片样品，结合1.5毫升超声的ð萃取液用抗体:珠缀合物在1.5ml或2ml管。 </li><li>以9速度设定孵育管上的微型管旋转过夜（大约16小时）在4℃。检查几个分钟后，以确保样本不漏，并适当混合。 </li></ol>第2天:沉淀洗和树脂上的酶反应 8.沉淀洗注意:要尽量减少交叉污染，简单地说每次冲洗之间旋转管。对于第一个愿望，改变每个样品之​​间的技巧。在随后的洗涤，同样尖端可以使用，只要它没有碰珠。请参阅有关正确木片水洗方向附加信息部分。 <ol><li>非常简要地旋转用离心管（〜500 XG 3秒）以收集液体在帽和在磁性架放置1分钟。同时仍然在磁性架，吸提取物。 </li><li>添加0.75毫升RIPA缓冲液到每个管中。卸下磁架管和颠倒几次混合。更换磁架及吸上清管。重复7倍。 </li><li>加0.75毫升的10mM Tris盐酸（pH7.5）中，以每管。卸下磁架管和颠倒几次混合。更换磁架及吸上清管。重复2倍。 </li><li>吸最后一次洗涤后，进行抛光。 注意:在部分9.3，10.3，11.3，12.3，13.3，14.3每次温育反应，和15.3，旋管短暂地方针对磁性机架1分钟和抽吸上清液后。洗2次以0.75毫升RIPA缓冲液和2×0.75 ml的三盐酸（pH7.5）中。 </li></ol> 9.抛光反应<ol><li>填写抛光主结构计算（ 表11）。使冰1.5mL管中混合抛光。吸取混合。 </li><li>最后的沉淀洗被吸入后，立即加入，同时还对磁架的抛光组合，以每个样品树脂的50微升。温育30分钟的样品在3 XG在一个恒温30℃。 </li><li>作为吸气8.4节之后的说明中描述最后一次洗涤后，前往A-拖尾。 </li></ol> 10.尾反应<ol><li>填写A尾主结构计算（ 表12）。使冰上1.5 ml管A尾组合。吸取混合。 </li><li>抛光部后，立即对磁架加入50微升A尾组合的每个样品的树脂，同时还。在一个恒温在3 XG孵育样品30分钟在37℃。 </li><li>作为吸气8.4节之后的说明中描述最后一次洗涤后，继续P7适配器结扎。 </li></ol> 11. P7适配器连接反应<ol><li>填写结扎主结构计算（ 表13）。使连接混合物在1.5ml管冰。移液器混合。 </li><li>紧接着A尾部分，添加48微升P7结扎预混液和2微升不同的适配器指数tO的同时仍然在磁架每个样品树脂。在恒温在3 XG孵育样品2小时在25℃。 注意:使用针对每个样本的不同的索引的将允许更多的样本，以在一个单一的流动池进行测序。 </li><li>作为吸气8.4节之后的说明中描述最后一次洗涤后，前往Φ-29尼克修复。 </li></ol> 12.披29尼克修复反应<ol><li>填写Φ-29主结构计算（ 表14）。使冰1.5mL管中Φ-29组合。吸取混合。 </li><li>紧接着P7结扎部分，添加50微升Φ-29组合的每个样品的树脂，同时还对磁架。在一个恒温在3 XG孵育样品20分钟，在30℃。 </li><li>作为吸气8.4节之后的说明中描述最后一次洗涤后，进行激酶反应。 </li></ol> 13.激酶反应<ol><li>填写激酶主结构计算（ <s仲&gt;表15）。让激酶混合物在1.5ml管冰。吸取混合。 </li><li>紧接着Φ-29部分，添加50微升激酶的同时仍然对磁架混合每个样品树脂。在一个恒温在3 XG孵育样品20分钟在37℃。 </li><li>作为吸气8.4节之后的说明中描述最后一次洗涤后，前往LAMBDA核酸外切酶反应。 </li></ol> 14. LAMBDA核酸外切酶反应<ol><li>填写LAMBDA核酸外切酶预混液计算（ 表16）。让LAMBDA核酸外切酶混合在1.5ml管冰。吸取混合。 </li><li>紧接着激酶部分，添加50微升LAMBDA核酸外切酶，同时仍对磁架混合每个样品树脂。在一个恒温在3 XG孵育样品30分钟在37℃。 </li><li>作为吸气8.4节之后的说明中描述最后一次洗涤后，前往RecJ˚F反应。 </li></ol> 15. RecJ˚F核酸酶反应<ol><li>填写RecJ˚F主结构计算（ 表17）。使冰1.5mL管中RecJ˚F组合。吸取混合。 </li><li>紧接着LAMBDA核酸外切段，加入50微升RecJ˚F组合的每个样品的树脂，同时还对磁架。在一个恒温在3 XG孵育样品30分钟在37℃。 </li><li>作为吸气8.4节之后的说明中描述最后一次洗涤后，进行芯片洗脱。 </li></ol> 16.洗脱和交联逆转<ol><li>制备主混合物:样本数x 1.1×1.5毫升管（200微升沉淀洗脱缓冲液+ 1微升20毫克/毫升蛋白酶K）。加入200μl的ChIP洗脱缓冲液+蛋白酶K预混每个样品树脂。 </li><li>在3 XG孵育样品过夜（约16小时），在65℃的恒温带热盖，以防止结露。 </li></ol>第3天:DNA提取离子和适配器结扎 17.洗脱和交联逆转（续） <ol><li>在65℃孵育过夜后，旋转简要样品收集冷凝水。在1分钟的磁架的地方样本。 </li><li>转移200μl的上清含有200微升TE缓冲液（pH7.5）中新的1.5毫升管。 </li></ol> 18.酚氯仿异戊醇（PCIAA）提取<ol><li>异戊醇添加400μl的苯酚氯仿至每个样品。涡旋混合20秒。 </li><li>在室温下（RT）20000 xg离心离心10分钟。仔细325微升上层水层的转移至新管。 注:请注意不要任何有机（下层）转移到新管。 </li><li>添加1/10容量的3M醋酸钠（pH值5.5）和1μl20mg / ml的糖原向每个样品。对于多个样本，准备一个主结构。 </li><li>添加3体积冰冷的100％乙醇，以每个样品。涡流混合。在-80℃孵育15分钟。 </li><li>离心机以20,000 xg离心在4℃15分钟。 小心倒出上清，确保不打扰颗粒。 </li><li>轻轻地加入500μl新鲜的冰冷的70％乙醇，并确保不打扰颗粒。离心机以20,000 xg离心在4℃5分钟。小心倒出上清液。 </li><li>干燥小丸，以用于在45℃的速度真空大约20分钟（或直至干燥）。 注意:这是在协议的暂停点。干燥DNA沉淀可以储存在-20℃。 </li><li>在10微升的DDH 2 O吸取悬浮粒料反复过其中的DNA丸状，即使将干燥的粒料不可见的区域。 </li><li>转移10微升各样品至一个新鲜0.3毫升PCR管或8架，这取决于样品的数目。 </li></ol> 19. P7引物延伸反应<ol><li>填写P7引物延伸主结构计算（ 表18）。使扩展混合在1.5ml管冰。吸取混合。 </li><li>添加扩展混合的10微升（减Φ-29聚合酶）每10微升样品。吸取混合。 </li><li>使用程序（ 表19）到退火的引物与模板直至30℃的热循环运行的样品"持有"步骤。 </li><li>在30℃时加入1μlΦ-29聚合酶"持有"的程序步骤。吸取混合。恢复计划（ 表19）的其余部分。 </li></ol> 20.尾反应<ol><li>填写A尾主结构计算（ 表20）。使冰上1.5 ml管A尾组合。吸取混合。 </li><li>加10μlA尾组合的每个样品。吸取混合。 </li><li>在热循环仪，孵育样品30分钟，在37℃，然后在75℃热灭活20分钟。 </li></ol> 21. P5适配器连接反应<ol><li>费尔L out输出P5接头连接主结构计算（ 表21）。使连接混合物在1.5ml管冰。吸取混合。 </li><li>加入20微升P5结扎混合每个样品。吸取混合。 </li><li>在热循环仪，孵育样品在25℃下2小时。 </li></ol> 22.珠清理注:请参阅制造商的详细信息资料部分上珠清理。 <ol><li>每50微升样品转移到一个新的1.5 ml管。 </li><li>悬浮在15速度设定清理上的微型管旋转体珠在室温下15分钟。不要让珠吸取之前结清。 </li><li>添加60微升清理珠样品（1.2:1清理珠粒采样比率是至关重要的，除去的DNA短于200个碱基对）。移液管混合20秒。 </li><li>悬浮在15速度设定清理上的微型管旋转体珠在室温下3分钟。旋转简要管。 </li><li>放置管上1分钟磁性机架。吸管关闭并丢弃上清液。 注意:不要用负压吸引在这一部分，因为它会吸了清理珠。 </li><li>加入400微升新鲜制备的RT 70％的乙醇，以每个样品，而他们上磁性机架上。不要混在一起。吸上清。重复2倍。 </li><li>干燥该清理珠在室温10分钟。珠的颜色会变成深到浅棕色和非常小的裂缝，当珠粒干将在树脂粒料可见。 </li><li>为了洗脱DNA，添加40微升的10mM的Tris（pH7.5）中。彻底吸管混合20秒。 </li><li>放置1分钟磁性机架样本。慢慢地吸取和36微升洗脱液直接转移到0.3毫升PCR管。 </li><li>在-20℃直接进行PCR反应或储存洗脱液。 </li></ol>第4天:PCR和凝胶分析 23. PCR <ol><li>填写PCR主混合计算（ 表22）。使PCR混合物。吸取轻轻混合，以避免气泡。 </li><li>加入14微升PCR的组合，以每36微升DNA样本。吸取混合。保持样品在冰上，直到准备把在热循环仪。 </li><li>对于阳性PCR对照，包括预先制备文库。对于阴性PCR对照，包括水。在热循环运行的样品使用PCR程序（ 表23）。 </li></ol> 24.凝胶制备，DNA切除和可视化<ol><li>彻底清洁和冲洗，用去离子水适当大小的凝胶盒。制备1.5％琼脂糖凝胶（含有0.5mg / ml溴化乙锭）配有可容纳60微升厚，宽孔梳。 注:溴化乙锭（乙锭）是一种致癌物质，必须小心处理。 </li><li>降速PCR样品管简单地收集冷凝。 </li><li>加入1/5体积的6倍二甲苯DNA染料的结合到样品。在DNA染料，因为它迁移在同一位置作为样品的DNA不使用溴酚蓝。 </li><li>加载整个样本INTØ每孔，最好有样本之间的空井。 注意:库具有相同的索引不能在同一凝胶上运行。 </li><li>负载7微升样本的两侧100bp的阶梯。在140 V代表约30-45分钟运行凝胶，直到阶梯溴苯酚染料是¾一路下滑的凝胶。 </li><li>图像，并在低UV设置可视上的透射凝胶。切下含有的DNA片段琼脂糖200的部分 - 500基点。要非常小心，以避免切割出运行在125 bp的适配器二聚体带。 </li><li>各切出凝胶切片放入15毫升管。记录净重凝胶片。直接在管道写这个重量。 </li><li>图像，保存和注释切除凝胶，以确认正确的尺寸范围内选择。 </li></ol> 25.凝胶纯化<ol><li>根据制造商的说明从切下的凝胶切片中回收DNA，并作以下修改。 </li><li>摇着溶解的凝胶片在室温在摇动平台。这将需要约20分钟使其溶解。 </li><li>以获得高度纯化的DNA，洗柱用0.5ml缓冲液QG溶解后凝胶已通过柱。 </li><li> PE缓冲液洗涤后，让列坐在RT 2 - 5分钟。在室温13000 xg离心旋转1分钟。 </li><li>为了留出空间PCR反应预混液，用40微升EB缓冲液洗脱样品到一个新的1.5 ml管。 </li></ol> 26. DNA定量<ol><li>根据制造商的说明（ 表24）制备的样品。测量样品中的光管中指定的仪器。 </li><li>一旦沉淀-外库量化，提交用于测序使用测序逐合成化学16，它与在此协议的DNA适配器相兼容的平台上。 注意:通常情况下，2微升样品是足够的量化，但更多的，如果试样的浓度低可能是必要的。 DNA产生典型50和200纳克之间光年范围内。 </li><li>量化后，店内样品在-20℃。 </li></ol>

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The authors have nothing to disclose.

<html>我们提出了一个功能基因组学的协议，以确定染色质以接近碱基对的分辨率相互作用的蛋白质在一个不带偏见，全基因组的方式精确绑定位置。实现近基部对映射解决最关键的一步就是芯片富集DNA的核酸外切酶处理而免疫沉淀留在磁性树脂。从表面上看，蛋白质复合物有可能阻断体内的任何给定的亚单位（如染色质重塑复合物或核小颗粒）的足迹。然而，如以前报道10中 ，由于甲醛是一种低效的交联剂，它变得越来越不可能，一个复杂的多个亚基将交联的DNA，并在同一轨迹彼此在相同的细胞。因此， 在蛋白质复合物的个别亚基，如核的个体组蛋白亚基体内足迹，可以带芯片-外。 T"&gt;芯片外型最显着的优点是其近碱基对分辨率和低背景，超高分辨率使得那些目前还无法与任何其他的方法来对基因组范围内进行了详细的结构及空间的见解。在另一方面，芯片外的主要限制是，这是一个在技术上具有挑战性的分子生物学方法来掌握，另外，该芯片步骤的一般性的限制也适用于沉淀-外（ 例如 ，商用抗体可用性和特异性簇，表位可访问性，和细胞的相对大量的需要）。常用的缺陷包括低质量的超声处理提取物，使用非沉淀级抗体，而不是保持在冰上的样品尽可能。因此，超声处理条件必须小心地优化各验证为先前抗体描述18，以避免有害影响，这些参数可能对实验结果。 据，直到...为止一个转录因子的目标测序深度，我们通常会瞄准约2000万唯一对准读取。因为聚合酶II和组蛋白修饰更广泛分布，我们的目标为30至5000万的读取。要注意的是，因为沉淀 - 外具有比芯片起背景基本上更小，更少的读取，需要实现类似测序深度是很重要的。 芯片外技术现在，尽管其技术上的挑战被广泛采用，如在原始协议鲁棒变化继续出版17,19,20。特别是，一种变体，可能为困难证明是有益的沉淀的蛋白质被称为的ChIP-关系，它使用一个单一的连接步骤，以增加文库制备20的效率更高。总之，芯片外是在全球范围内染色质相互作用蛋白的超高分辨率测绘的越来越多的利用和强大的方法论。作为市售的ChIP同类蚂蚁的列表ibodies继续增长，芯片 - 外方法的未来的应用程序将在映射未知基因调控网络被引导到理解在超高分辨率的细胞的分子电路。此外，芯片外很可能会进一步完善和调整以接近碱基对的决议，足迹体内蛋白质的RNA相互作用。

染色质免疫沉淀（ChIP）是一种强有力的方法通过对与在活细胞中给定蛋白质相互作用的DNA片段选择性富集来研究基因调控机制。的ChIP富集的DNA片段的检测方法已演变随着技术的改进，从检测单一位点（标准芯片定量PCR）的对寡核苷酸芯片（芯片芯片），以高通量测序（芯片起）1杂交。虽然芯片起已经看到了广泛的应用，染色质异质性和非特异性DNA的相互作用，阻碍了数据质量，导致误报和不准确的映射。为了规避这些限制，弗兰克普格博士开发芯片外方法2。沉淀 - 外的显着特点是，它包括一个5&#39;至3&#39;外切核酸酶，从而有效地足迹转录因子结合的位置。其结果，芯片外的方法实现了更高的分辨率，detectio的更大的动态范围N，和较低的背景噪音。 虽然沉淀外型更加技术上具有挑战性的掌握比芯片起，它被广泛地研究的目的使用不同的生物系统3-8获得独特的超高分辨率的见解采纳。实际上，芯片外已成功地应用于细菌，酵母，小鼠，大鼠和人的细胞系统。作为证据原则，芯片外原本用于识别的精确结合基序的酵母转录一把因素2。也用于在酵母中的技术，研究了转录预起始复合物的组织，并破译各种组蛋白9,10 subnucleosomal结构。最近，我们利用的ChIP-外解决相邻TFIIB和Pol II结合在促进人的事件，并表明广泛转录分歧源于不同的起始复合物11。 这里提出的工作流程最优化和streamlined用于哺乳动物的ChIP -外（ 图1）。首先，住伯或组织培养细胞用甲醛处理通过共价交联的体内蛋白-DNA相互作用保存。将细胞裂解和染色质剪切至〜100 - 500碱基对大小的片段。沉淀然后选择性富集为交联到感兴趣的蛋白质的DNA片段。在这点上，芯片起库通常制备，这固有地限制了检测分辨率到几百个碱基对的平均片段大小。然而，芯片外克服通过调整左和右5所述蛋白质-DNA交联位点以拉姆达核酸外切酶的"DNA边界此限制。测序文库，然后从外切核酸酶消化的DNA构建了如下详述。所得嵌套5&#39;边界代表蛋白质-DNA相互作用（ 图1，步骤14）的体内足迹，并且通过高通量测序进行检测。 Alt键霍夫芯片外的方法比芯片起更多的参与，多数步骤之间的转换需要简单洗珠，最大限度地减少样品损失和实验的可变性。重要的是，由于沉淀外型是芯片起，即成功的芯片起也应该是成功的为片外的任何样品的改良版本。 在从芯片起一个根本不同的数据结构的ChIP外型结果蛋白-DNA相互作用的体内足迹。虽然通用芯片起呼叫者可以应用到芯片外的数据，以获得最精确的峰值呼叫，我们建议专门为片外数据心目中的独特的结构设计，生物信息学工具。这些措施包括Genetrack，创业板，MACE，Peakzilla和ExoProfiler 12-15。

<table><tbody><tr><td>37% formaldehyde, methanol free, 10 x 10 ml ampules</td><td>ThermoFisher Scientific</td><td>28908</td><td>Section 3</td></tr><tr><td>Complete Protease Inhibitor cocktail (CPI)</td><td>Roche Life Science</td><td>11873580001</td><td>Sections 4, 8</td></tr><tr><td>Bioruptor sonicator</td><td>Diagenode</td><td>UCD200</td><td>Section 5</td></tr><tr><td>15 ml polystyrene tubes</td><td>BD Falcon</td><td>352095</td><td>Section 5</td></tr><tr><td>MagSepharose Protein G Xtra beads</td><td>GE Healthcare</td><td>28-9670-66</td><td>Section 6</td></tr><tr><td>DynaMag-1.5 ml Side Magnetic Rack</td><td>Invitrogen</td><td>12321D</td><td>Sections 6-17, 22</td></tr><tr><td>Mini-Tube Rotator</td><td>Fisher Scientific</td><td>05-450-127</td><td>Sections 7, 22</td></tr><tr><td>T4 DNA Polymerase</td><td>New England BioLabs</td><td>M0203</td><td>Section 9</td></tr><tr><td>DNA Polymerase I, Klenow</td><td>New England BioLabs</td><td>M0210</td><td>Section 9</td></tr><tr><td>10x NEBuffer 2 (10x Reaction Buffer 2)</td><td>New England BioLabs</td><td>B7002</td><td>Sections 9-11, 13, 15, 20</td></tr><tr><td>Thermomixer C</td><td>Eppendorf</td><td>5382</td><td>Sections 9-16</td></tr><tr><td>ATP</td><td>Roche Life Science</td><td>010419979001</td><td>Sections 9, 11, 13</td></tr><tr><td>dNTPs</td><td>New England BioLabs</td><td>N0447</td><td>Sections 9, 12, 19, 23</td></tr><tr><td>T4 Polynucleotide Kinase</td><td>New England BioLabs</td><td>M0201</td><td>Sections 9, 13</td></tr><tr><td>dATP</td><td>New England BioLabs</td><td>N0440</td><td>Sections 10, 20</td></tr><tr><td>Klenow 3&#039;-5&#039; Exo Minus</td><td>New England BioLabs</td><td>M0212</td><td>Sections 10, 20</td></tr><tr><td>T4 DNA ligase&amp;nbsp;</td><td>New England BioLabs</td><td>M0202</td><td>Sections 11, 21</td></tr><tr><td>&amp;Phi;-29 DNA Polymerase</td><td>New England BioLabs</td><td>M0269</td><td>Sections 12, 19</td></tr><tr><td>10x &amp;Phi;-29 Buffer</td><td>New England BioLabs</td><td>B0269</td><td>Sections 12, 19</td></tr><tr><td>Lambda Exonuclease</td><td>New England BioLabs</td><td>M0262</td><td>Section 14</td></tr><tr><td>10x Lambda Buffer</td><td>New England BioLabs</td><td>B0262</td><td>Section 14</td></tr><tr><td>RecJ&lt;sub&gt;f&lt;/sub&gt; Exonuclease</td><td>New England BioLabs</td><td>M0264</td><td>Section 15</td></tr><tr><td>Proteinase K</td><td>Roche Life Science</td><td>03115828001</td><td>Section 16</td></tr><tr><td>Glycogen</td><td>Roche Life Science</td><td>010901393001</td><td>Section 18</td></tr><tr><td>10x T4 Ligase Buffer</td><td>New England BioLabs</td><td>B0202</td><td>Section 21</td></tr><tr><td>AMPure XP (clean up) beads&amp;nbsp;</td><td>Beckman Coulter</td><td>A63881</td><td>Section 22</td></tr><tr><td>Q5 Hot Start DNA Polymerase&amp;nbsp;</td><td>New England BioLabs</td><td>M0493</td><td>Section 23</td></tr><tr><td>5x Q5 Buffer (5x PCR Buffer)</td><td>New England BioLabs</td><td>B9027</td><td>Section 23</td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>Qiagen</td><td>28704</td><td>Section 25</td></tr><tr><td>Qubit Fluorometer</td><td>Invitrogen</td><td>Q33216</td><td>Section 26</td></tr><tr><td>Optical Clear Qubit tubes&amp;nbsp;</td><td>Invitrogen</td><td>Q32856</td><td>Section 26</td></tr><tr><td>Qubit dsDNA High Sensitivity Assay kit</td><td>Invitrogen</td><td>Q32851</td><td>Section 26</td></tr></tbody></table>

the chip-exo method: identifying protein-dna interactions with near base pair precision

染色质免疫沉淀（ChIP）是表观遗传学和基因调节的隔离特定蛋白质-DNA相互作用领域不可缺少的工具。沉淀耦合到高通量测序（芯片起）通常用于确定与染色质相互作用的蛋白质的基因组位置。然而，芯片起是由几百个碱基对相对低的映射分辨率和高背景信号受到阻碍。芯片外的方法是芯片起的改良版本对分辨率和噪声大大提高。芯片外方法论的主要区别是:在图书馆准备的工作流程，有效足迹左侧的lambda核酸外切酶消化的引入和蛋白质-DNA交联网站的权5&#39;DNA边界。然后芯片外库进行高通量测序。所得到的数据可以被利用，以提供独特的和超高分辨率分析上市功能组织ÒF中的基因组中。在这里，我们描述了我们优化和精简了哺乳动物系统和新一代测序边合成平台芯片外的方法。

Watch this Scientific Journal Video about The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision at JoVE.com

ChIP-exo 方法：以近碱基对精度识别蛋白质-DNA 相互作用 |生物学 |视频

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

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 ...

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DNA Structure and Function

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16.5K 次观看.  Vanderbilt University. 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.

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

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

与ChEC-seq的蛋白质-DNA相互作用的全基因组映射<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

科研

遗传学

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein interactions in yeast. It allows analysis of a target region of interest without the need for prior knowledge about likely proteins bound to the target region. This, in theory, allows HyCCAPP to be used to analyze any genomic region of interest, and it provides sufficient flexibility to work in different cell systems. This method is not meant to study binding sites of known transcription factors, a task better suited for Chromatin Immunoprecipitation (ChIP) and ChIP-like methods. The strength of HyCCAPP lies in its ability to explore DNA regions for which there is limited or no knowledge about the proteins bound to it. It can also be a convenient method to avoid biases (present in ChIP-like methods) introduced by protein-based chromatin enrichment using antibodies. Potentially, HyCCAPP can be a powerful tool to uncover truly novel DNA-protein interactions. To date, the technology has been predominantly applied to yeast cells or to high copy repeat sequences in mammalian cells. In order to become the powerful tool we envision, HyCCAPP approaches need to be optimized to efficiently capture single-copy loci in mammalian cells. Here, we present our adaptation of the initial yeast HyCCAPP capture protocol to human cell lines, and show that single-copy chromatin regions can be efficiently isolated with this modified protocol.

This is a method to identify novel DNA-interacting proteins at specific target loci, relying on sequence-specific capture of crosslinked chromatin for subsequent proteomic analyses. No prior knowledge about potential binding proteins, nor cell modifications are required. Initially developed for yeast, the technology has now been adapted for mammalian cells.

蛋白质组学与染色质相关蛋白杂交捕获对哺乳动物细胞的适应性研究

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein ...

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 ...

CD Spectroscopy to Study DNA-Protein Interactions

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent advancements in CD spectroscopy have enabled the study of DNA-protein interactions and conformational dynamics of DNA in different microenvironments in detail for a better understanding of transcriptional regulation in vivo. The area around a potential transcription zone needs to be unwound for transcription to occur. This is a complex process requiring the coordination of histone modifications, binding of the transcription factor to DNA, and other chromatin remodeling activities. Using CD spectroscopy, it is possible to study conformational changes in the promoter region caused by regulatory proteins, such as ATP-dependent chromatin remodelers, to promote transcription. The conformational changes occurring in the protein can also be monitored. In addition, queries regarding the affinity of the protein towards its target DNA and sequence specificity can be addressed by incorporating mutations in the target DNA. In short, the unique understanding of this sensitive and inexpensive method can predict changes in chromatin dynamics, thereby improving the understanding of transcriptional regulation.

The interaction of an ATP-dependent chromatin remodeler with a DNA ligand is described using CD spectroscopy. The induced conformational changes on a gene promoter analyzed by the peaks generated can be used to understand the mechanism of transcriptional regulation.

CD光谱学研究DNA-蛋白质相互作用

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent ...

生物化学

Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14. 
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8. 
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.

Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.

配对末端标签测序（嘉PET）测绘染色质相互作用和了解转录调控染色质相互作用分析

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such ...

生物学

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

的蛋白质相互作用的合作伙伴在哺乳动物细胞中使用SILAC-免疫定量蛋白质组学鉴定

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...

DNA-affinity-purified Chip (DAP-chip) Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of limited use in exploring bacterial two component regulatory systems with uncharacterized activation conditions. Such systems regulate transcription only when activated in the presence of unique signals. Since these signals are often unknown, the in vitro microarray based method described in this video article can be used to determine gene targets and binding sites for response regulators. This DNA-affinity-purified-chip method may be used for any purified regulator in any organism with a sequenced genome. The protocol involves allowing the purified tagged protein to bind to sheared genomic DNA and then affinity purifying the protein-bound DNA, followed by fluorescent labeling of the DNA and hybridization to a custom tiling array. Preceding steps that may be used to optimize the assay for specific regulators are also described. The peaks generated by the array data analysis are used to predict binding site motifs, which are then experimentally validated. The motif predictions can be further used to determine gene targets of orthologous response regulators in closely related species. We demonstrate the applicability of this method by determining the gene targets and binding site motifs and thus predicting the function for a sigma54-dependent response regulator DVU3023 in the environmental bacterium Desulfovibrio vulgaris Hildenborough.

DNA-affinity-purified Chip DAP-chip Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

This video article describes an in vitro microarray based method to determine the gene targets and binding sites for two component system response regulators.

DNA的亲和纯化的芯片（DAP芯片）方法来确定目标基因的细菌双组分调控系统

In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).

药物与DNA相互作用的细胞全基因组映射与COSMIC（小分子交联，染色质隔离）

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

细菌、酵母和人类细胞中瞬态形成的分子茶龙组件的原位监测

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is essential in revealing cellular events associated with the recruitment and assembly of DNA repair complexes at the site of DNA damage. In the last few years, several powerful tools have been developed to induce site-specific DNA damage. Moreover, novel seminal techniques allow us to study these processes at the single-cell resolution level using both fixed and living cells. Although these techniques have been used to study various biological processes, herein we present the most widely used protocols in the field of DNA repair, Fluorescence Immunostaining (IF) and Chromatin Immunoprecipitation (ChIP), which in combination with endonuclease-based site-specific DNA damage make it possible to visualize and quantify the genomic occupancy of DNA repair factors in a directed and regulated fashion, respectively. These techniques provide powerful tools for the researchers to identify novel proteins bound to the damaged genomic locus as well as their post-translational modifications necessary for their fine-tune regulation during DNA repair.

This article introduces essential steps of immunostaining and chromatin immunoprecipitation. These protocols are commonly used to study DNA damage-related cellular processes and to visualize and quantify the recruitment of proteins implicated in DNA repair.

可视化和量化基于内分泌酶的站点特定DNA损伤

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is ...