We thank Dr. Bas van Steensel for providing the DamID mammalian expression vectors. We thank Yale Center for Genome Analysis and the Genomics Core in Yale Stem Cell Center for advice on preparing NGS libraries and implementing high throughput DNA sequencing. This work was supported by the startup funding from Yale School of Medicine, a Scientist Development Grant from American Heart Association (12SDG11630031) and a Seed Grant from Connecticut Innovations, Inc. (13-SCA-YALE-15).

1.产生和融合蛋白和自由大坝蛋白的表达<ol><li> 感兴趣进入DamID载体克隆蛋白。 <ol><li>根据制造商的方案扩增使用所需的高保真DNA聚合酶的兴趣点（POI）的蛋白质和合适的引物的cDNA的。实验确定最佳扩增条件，以确保插入适当的放大。 </li><li>运行一个琼脂糖凝胶并根据制造商的方案纯化的POI扩增的cDNA由凝胶提取试剂盒。 </li><li>克隆的cDNA的POI成采用BP克隆酶II根据制造商的协议的pDONR201载体中。 </li><li>根据制造商的方案从供体载体导入使用LR克隆酶II的pLGW-RFC1-V5-EcoDam目的载体或pLGW-EcoDam-V5-RFC1目的向量27克隆的POI中的cDNA，这取决于熔融的POI的期望的方向到N末端或次大肠电子C-末端大肠杆菌DNA甲基腺嘌呤（EcoDam）27。 </li><li>验证通过测序，克隆的cDNA具有一个正确的顺序和形式框内融合到EcoDam。 </li></ol></li><li> 生成慢病毒股票。 <ol><li>生成慢的股票采用慢病毒表达系统表达大坝-V5-POI和V5-大坝（从pLGW-V5-EcoDam矢量27）。根据制造商的方案使用正转染过程。 <ol><li>使用293T细胞和脂质转染根据制造商的方案进行转染，以产生慢病毒股票。 </li><li>包括0.45微米的PVDF过滤步骤。 </li></ol></li></ol></li><li> 感染细胞的慢病毒。 <ol><li>感染（0天）的前一天，使用相同生长培养基通过贴壁细胞在合适的生长培养基中培养该细胞类型向6孔组织培养板无抗生素到实现对感染当天50％汇合。位置细胞在37℃培养箱。 </li><li>感染（第1天）的一天，从取出2冻存管两个大坝-V5-POI和V5-大坝慢病毒上清液-80℃冰箱，并装在一个37℃水浴解冻。 <ol><li>从细胞中除去生长培养基并用0.5ml新鲜的生长培养基替换不含抗生素。 </li><li>加入1毫升的解冻慢病毒向每个孔（2井用V5-坝和2井用坝-V5-POI）。不含抗生素加入1毫升生长培养基向剩下的2孔中，这将作为阴性对照。轻轻晃动6孔板混匀，早在37℃培养箱O / N。 </li></ol></li><li>感染（第2天）后一天，从细胞中除去病毒悬浮液和用2ml生长介质代替不含抗生素。将细胞早在37℃的培养箱中培养48小时。 </li></ol></li><li> 隔离基因组DNA。 <ol><li>从每个孔和德抽吸媒体用250微升0.05％胰蛋白酶-EDTA转速细胞。在37℃下2分钟。 </li><li>从每个孔1ml生长培养基和吸管细胞洗涤细胞脱盘入1.5ml微量离心管中。通过在200×g离心离心5分钟，在RT下沉淀细胞。 </li><li>在200×g离心在RT 2分钟洗涤沉淀的细胞用500μl的PBS，离心。 </li><li>重悬沉淀的细胞在200μlPBS中。 </li><li>根据制造商的方案分离的gDNA通过血液和组织试剂盒。洗脱的gDNA在200μl缓冲液AE和确定通过使用分光光度计测量的OD 260的浓度。 注:基因组DNA未感染的或模拟感染的细胞可以分离作为阴性对照。沉淀的gDNA以用于长期存储更高的浓度。 <ol><li>添加3倍体积的100％乙醇和0.1体积的3M乙酸钠（pH 5.5），并通过反相管4-6次混合。 </li><li>储存在-20°CO / N。 </li><li>离心机在16,000xg下克15分钟，在4℃。 </li><li>小心取出上清液。在4℃下进行5分钟，在16000×g离心洗粒料用70％（体积/体积）乙醇和离心机。 </li><li>小心除去乙醇并允许粒料空气干燥3分钟，在室温。 </li><li> T中10 E 0.1（pH值7.5）基因组DNA溶解到大约1微克/微升。池的gDNA从每个实验样品或阴性对照和测量的浓度。储存在-20℃。 </li></ol></li></ol></li></ol> 2.扩增出腺嘌呤甲基化的DNA片段<ol><li> 摘要基因组DNA与DPNI，只有切断腺嘌呤甲基化GATCs。 <ol><li>设置在冰上反应用2.5微克的gDNA，1微升10X缓冲液，0.5μl的DPNI（20U /微升）和用H 2 O充满至10微升的总体积。对于每一个基因组DNA样品，准备三种反应-一个没有DPNI（"无DPNI"，与0.5微升H 2 O的替代DPNI）和2瓦特第i个DPNI（"与DPNI"）。 </li><li>消化O / N在37℃和灭活DPNI在80℃下进行20分钟。 </li></ol></li><li> 结扎DamID适配器。 <ol><li>准备DamID适配器。 <ol><li>在H 2 O的悬浮每两个DamID适配器寡聚24到100微米的最终浓度。 </li><li>结合两种DamID适配器寡聚物等体积混合，并放置在密闭的管中。密封用Parafilm管，坐在机架并置于在90℃充满水的烧杯中。保持所述管的盖下面的水平（以避免水进入管），但低聚混合物的表面上方。 </li><li>让水冷却至室温，所以适配器退火缓慢。 </li><li>分装退火适配器（50μM），并存储在-20℃。 </li></ol></li><li>设置在冰上进行反应。在从2.1.2各管中，加入6.2微升H 2 0，2微升10X连接缓冲液，0.8微升50微米DamID广告aptors（ 在冰上解冻 ）和1μlT4DNA连接酶（5U /微升）。总体积为20微升。在这两个"与DPNI"管之一，用1微升的 H 2 O（"无连接酶"）代替连接酶注意，每个基因组DNA样品具有两个阴性对照- "无DPNI"和"无连接酶"。 </li><li>结扎O / N在16°C和灭活连接酶在65℃下10分钟。 </li></ol></li><li> 摘要DNA与DpnII销毁含有未甲基化GATCs片段。 <ol><li>设置在冰上进行反应。在从2.2.3各管中，加入24微升H 2 O的，5微升10X DpnII缓冲液和1微升DpnII（10U /微升）。总体积为50微升。 </li><li>消化在37℃进行2-3小时，并灭活DpnII在65℃下20分钟。 </li></ol></li><li> 放大腺嘌呤甲基化的DNA片段。 <ol><li>建立在冰上用5微升反应DpnII消化从2.3.2，5μ微升10x PCR缓冲液，12.5微升5微米DamID PCR引物24，4微升10毫米的dNTP混合，1微升50X聚合酶混合物和22.5微升H 2 O.总体积为50微升。 </li><li>运行的PCR如下:68℃，10分钟; 94℃下1分钟，65℃5分钟，68℃15分钟; 4个循环的94℃下1分钟，65℃1分钟，68℃，10分钟; 1分钟，65℃1分钟，68℃2分钟，17个循环的94℃。 </li><li>从在1％琼脂糖凝胶各反应分析5微升PCR产物。 PCR产物应显示为一个涂抹范围从0.2到2千字节（ 图2）。 "无DPNI"和"无连接酶"对照应该没有或明显较少的扩增。 </li><li>如果从步骤2.4.3结果是满意的，重复步骤2.4.1-2.4.3具有两个或三个反应的实验样品和对于每两个阴性对照中的一个反应。 </li><li>池和从相同的纯化的PCR产物使用PCR纯化试剂盒或固相可逆固定（SPRI）珠粒根据制造商的方案的实验样品。不要净化"不DPNI"或"无连接酶"的控制。 DNA洗脱缓冲液EB。 </li><li>通过使用分光光度计，其应该在0.1微克/微升或更高测量的OD 260测量纯化的DNA的浓度。收集至少10微克的DNA各样品。如果使用PCR纯化试剂盒，净化每50微升PCR产物有一列，洗脱在30微升缓冲EB，集中洗脱液。 </li></ol></li><li> 摘要DNA与DpnII防止DamID PCR引物被测序。 <ol><li>建立从2.4.6在冰上反应用5微克纯化的DNA，5微升10X DpnII缓冲液，1微升DpnII（10U /微升）和用H 2 O充满至50微升的总体积。准备两个或三个反应各样品。 </li><li>消化在37℃进行2-3小时和灭活DpnII在65℃下20分钟。 </li><li>池以及从与PCR纯化试剂盒或SPRI珠粒同一样品根据生产商的方案纯化消化物。 DNA洗脱缓冲液EB。 </li><li>测量纯化的DNA的浓度应为约0.06微克/微升或更高。收集至少6微克的DNA各样品。如果使用PCR纯化试剂盒，净化每50有一列微升消化，洗脱在30微升缓冲EB，集中洗脱液。 </li></ol></li></ol> 3.文库制备的高通量测序<ol><li> 片段DNA <ol><li>实验确定适当的消化时间为每个新一批双链DNA Fragmentase的。由于酶的活性会降低随着时间的推移，执行新的实验前重复测试。要保存DNA从2.5.4实际的碎片，使用纯净甲基PCR产物2.4.6或以前的experime额外的DNA的nts在此步骤。 <ol><li>设置了主混合物与6微克的DNA，12微升10X Fragmentase缓冲器和用H 2 O充满至114微升的总体积。 </li><li>涡Fragmentase股票小瓶3秒，加入6微升到主结构和涡主结构，持续3秒。总体积为120微升。 </li><li>分装20微升的主结构，以每5个新管。孵育所有6的管在37℃进行5至55分钟，在10分钟的增量。添加5微升的0.5M EDTA停止反应。 </li><li>分析12.5微升消化（0.5微克DNA），每个反应以及0.5微克琼脂糖凝胶上的DNA未消化（图3）。确定多数涂抹消化约0.2 kb的所需的最少时间（T 0.2kb）。选择（包括5分钟和T 0.2kb）与实际的碎片等额递增5分钟和T 0.2kb的6持续时间。 </li></ol></li><li>设置实际fragmenTATION在3.1.1.1-3.1.1.3所述。孵育反应在37℃下，在3.1.1.4所确定的持续时间。 </li><li>池6的反应和纯化用PCR纯化试剂盒或根据制造商的方案SPRI珠粒的摘要。稀释成51微升缓冲EB DNA。最后的洗脱液〜50微升。 </li></ol></li><li> 零碎的DNA修复结束 <ol><li>设置了从3.1.3洗脱液，25微升H于冰上反应2 O，10微升10X T4 DNA连接酶缓冲液，10毫摩尔ATP，4微升的10mM dNTP混合物，5微升T4 DNA聚合酶（3U /微升） ，1微升Klenow酶DNA聚合酶（5U /微升）和5微升T4多核苷酸激酶。总体积为100微升。用移液拌匀。避免泡沫和气泡。 </li><li>孵育在20℃下30分钟。 </li><li>净化用PCR纯化试剂盒或根据制造商的方案SPRI珠粒的DNA。稀释成33微升缓冲EB DNA。最后的洗脱液〜32＃181，L。 </li></ol></li><li> 加入"A"出挑 <ol><li>建立从3.2.3在冰上反应与洗脱液，5微升10×Klenow酶缓冲液，10微升1 10mM的dNTP，和3微升Klenow酶（3&#39;→5&#39;外切）（5U /微升）。总体积为50微升。用移液拌匀。避免泡沫和气泡。 </li><li>在37℃下进行30分钟。 </li><li>净化用PCR纯化试剂盒或根据制造商的方案SPRI珠粒的DNA。稀释成22微升缓冲EB DNA。最后的洗脱液〜21微升。 </li></ol></li><li> 结扎测序适配器 <ol><li>准备测序接头28。 <ol><li>重悬每个适配器寡聚至100μM的在10mM Tris-Cl（上pH值7.8），0.1毫摩尔EDTA（pH8.0）中和50mM NaCl的浓度。 </li><li>混合的通用适配器28和索引适配器28等体积。 </li><li>退火适配器用下面的热循环仪程序:95℃，2分钟; 140个循环30秒开始在95℃和0.5℃的每个周期降低;保持在4℃。 </li><li>分装适配器并储存在-20℃。 </li></ol></li><li>设置在冰上从3.4.1.4和2.5微升T4 DNA连接酶的反应从3.3.3洗脱液，25μl的2×连接缓冲液，1.5微升50μM的退火测序接头（在冰上解冻）。用移液拌匀。避免泡沫和气泡。如果多重测序需要，使用不同的索引适配器每个样品。 </li><li>在室温下孵育1小时。 </li><li>净化用PCR纯化试剂盒或根据制造商的方案SPRI珠粒的DNA。稀释成24微升缓冲EB DNA。最后的洗脱液〜23微​​升。 </li></ol></li><li> 转换Y形适配器双链DNA，以便准确地确定DNA片段大小28 <ol><li>设置在冰上的反应与3.4.4洗脱液，12.5微升H 2 O的，1微升25μM的引物1 28，1微升25μM的引物2 28 1.5微升的10mM的dNTP，10微升5×PCR缓冲液和1μl的DNA聚合酶（1U /微升）。 </li><li>运行的PCR如下:95℃，3分钟; 5个循环的98℃15秒，63℃，30秒，72℃30秒; 72℃，1分钟; 4℃搁置。 </li><li>净化用PCR纯化试剂盒或根据制造商的方案SPRI珠粒的DNA。稀释成11微升缓冲EB DNA。最后的洗脱液〜10微升。 </li></ol></li><li> 大小选择库 <ol><li>制备用1×TAE缓冲液的2％琼脂糖凝胶。加入溴化乙锭（ 小心！），以500纳克/毫升的最终浓度时熔化TAE琼脂糖溶液冷却，以避免溴化乙锭的吸入。确保有足够的通道对所有样品，DNA梯和空车道。 </li><li>从3.5.3添加8微升6X样染料的洗出液。 </li> &lt;李&gt;混合1 kb的DNA加梯（1.0微克/微升），6X样染料和H 2 O以1:2的比例制备DNA梯:1:4。 </li><li>负载6微升DNA梯，从3.6.2在一个单独的车道，并与相邻的采样/梯子至少一个空巷的样本和另外6微升DNA阶梯，每一个。 </li><li>运行在120伏的凝胶60分钟。 </li><li>查看紫外透射凝胶（最小化的曝光时间，以UV）的。戴安全防护眼镜和面罩。确保该DNA梯中的至少一个具有适当间隔（足够用于切除3凝胶切片）300 bp和400bp的条带之间良好运行。窄间距增大切除多个凝胶切片的困难，而宽的间隔增大切凝胶切片的体积。 </li><li>使用新的手术刀或刀片用于每个通道。消费300和从每个车道的400bp之间的三个薄凝胶切片（图4），并将其放置在每一个微量离心管中。保持每个胶S的量虱尽可能低（&lt;100微升）。 </li><li>测量每个凝胶片（1微升凝胶重量约为1毫克）的量，加QG缓冲区6X体积和孵化在50℃。 </li><li> VORTEX的QG-凝胶混合物每2-3分钟，直到凝胶条完全溶解。加入异丙醇2倍凝胶体积和混合。 </li><li>从这个步骤，遵循凝胶提取试剂盒的协议。稀释成51微升缓冲EB DNA。最后的洗脱液〜50微升。 </li></ol></li><li> 丰富测序衔接修饰的DNA片段 <ol><li>优化PCR循环29的数目。 <ol><li>设置了从10年6月3日1微升洗脱液，1微升25μM的引物1 28，1微升25μM的引物2 28 7微升的 H 2 O和10微升的SYBR Green超在冰上进行反应。总体积为20微升。 </li><li>运行的qPCR如下:95℃，3分钟; 20个循环的95℃，30秒，63℃30秒，72℃，30秒，读板。 </li><li>分析用的qPCR分析软件数据，并确定所述定量循环（CQ）或阈值循环数（Ct）使用制造商的协议的每个样品。继续与具有Cq的/ CT≤14.使用样品的最大的Cq（Cq的0）减去1（上舍入到下一个较高的整数）作为最终的PCR循环数目（N PCR）。 </li><li>调整的DNA模板的量，使不同的样品将在运行的PCR循环数相同之后被放大到近似等量。使用8微升模板具有最高的Cq（Cq的0）的样品，并计算其它样品用以下公式的模板量: 第一卷 = 8×1.8 的Cq 我 -cq 0 </li></ol></li><li>设置PCR反应在冰上用从3.7.1.4计算出的模板卷:1微升25μM的引物1 28，1微升25μM的引物2 28 1.5微升的10mM的dNTP，10微升5×缓冲液，1μl的DNA聚合酶（1U /微升）和用H 2 O充满至50微升的总体积。 </li><li>运行的PCR如下:95℃45秒; ÑPCR循环 98℃（在3.7.1.3确定），15秒，63℃，30秒，72℃30秒; 72℃，1分钟; 4℃搁置。 </li><li>在2％琼脂糖凝胶（图5A）分析5μl的PCR产物。一个明显的"单一"带表明扩增的DNA片段是一个窄的长度范围内，并且该DNA库可以进行进一步的分析。 </li><li>重复一反应对选定的样品和从同一样品池扩增的DNA文库。 </li><li>净化测序选择的DNA库。 <ol><li>如果引物/二聚体适配器是不可见的3.7.4，净化用PCR纯化试剂盒或根据制造商的方案SPRI珠粒的DNA文库。稀释成21微升缓冲EB DNA。该最后的洗脱液〜20微升。如果用户的测序设施对洗脱缓冲液中，最终体积等的具体说明，相应的准备样品。 </li><li>如果底漆/适配器二聚体都清晰可见3.7.4，净化库如下。 <ol><li>从3.7.5负载DNA和DNA的阶梯在2％的预制琼脂糖凝胶。如3.7.6.1中所述，以减少体积或可以装入多个井的样品可被纯化。将琼脂糖凝胶在适当的电源系统。使凝胶运行15分钟。 </li><li>使用适当的透射，切出的期望的频带用干净的解剖刀/剃刀每个样品，然后将凝胶切片在1.5ml微量离心管中。 </li><li>根据制造商的方案用两个修饰使用凝胶提取试剂盒分离DNA:在一个热混合器孵育缓冲QG-凝胶混合物在37℃和1000转进行30分钟，并添加异丙醇2×凝胶卷。 </li></ol></li></ol></li></ol></li><li> 提交纯化的DNA库，测序设施。遵循所有设施的准则。 注意:由生物分析仪（图5B）一个质量分析，以确定确切的尺寸范围和DNA文库的浓度应进行测序之前。 </li></ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+targets+of+the+D.+melanogaster+DSXF+protein+and+the+evolution+of+sexual+development.">Direct targets of the D. melanogaster DSXF protein and the evolution of sexual development.</a> Development. 138, 2761-2771 (2011).</li><li>Germann, S., Gaudin, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+in+vivo+protein-DNA+interactions+in+plants+by+DamID,+a+DNA+adenine+methylation-based+method.">Mapping in vivo protein-DNA interactions in plants by DamID, a DNA adenine methylation-based method.</a> Methods Mol Biol. 754, 307-321 (2011).</li><li>Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+LHP1+protein+colocalizes+with+histone+H3+Lys27+trimethylation.">The Arabidopsis LHP1 protein colocalizes with histone H3 Lys27 trimethylation.</a> Nat Struct Mol Biol. 14, 869-871 (2007).</li><li>Orian, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+profiling,+DamID+and+the+emerging+landscape+of+gene+expression.">Chromatin profiling, DamID and the emerging landscape of gene expression.</a> Curr Opin Genet Dev. 16, 157-164 (2006).</li><li>Vogel, M. J., Peric-Hupkes, D., van Steensel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+in+vivo+protein-DNA+interactions+using+DamID+in+mammalian+cells.">Detection of in vivo protein-DNA interactions using DamID in mammalian cells.</a> Nat Protoc. 2, 1467-1478 (2007).</li><li>Greil, F., Moorman, C., van Steensel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DamID:+mapping+of+in+vivo+protein-genome+interactions+using+tethered+DNA+adenine+methyltransferase.">DamID: mapping of in vivo protein-genome interactions using tethered DNA adenine methyltransferase.</a> Methods Enzymol. 410, 342-359 (2006).</li><li>de Wit, E., Greil, F., van Steensel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+HP1+binding+in+Drosophila:+developmental+plasticity+and+genomic+targeting+signals.">Genome-wide HP1 binding in Drosophila: developmental plasticity and genomic targeting signals.</a> Genome Res. 15, 1265-1273 (2005).</li><li>. Illumina TruSeq adaptors &amp; PCR primers Available from: <a target="_blank" href="https://ethanomics.wordpress.com/chip-seq-library-construction-using-the-illumina-truseq-adapters/">https://ethanomics.wordpress.com/chip-seq-library-construction-using-the-illumina-truseq-adapters/</a> (2015)</li><li>. Optimization of PCR cycles for NGS Available from: <a target="_blank" href="https://ethanomics.wordpress.com/ngs-pcr-cycle-quantitation-protocol/">https://ethanomics.wordpress.com/ngs-pcr-cycle-quantitation-protocol/</a> (2015)</li><li>Bernstein, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NIH+Roadmap+Epigenomics+Mapping+Consortium.">The NIH Roadmap Epigenomics Mapping Consortium.</a> Nat Biotechnol. 28, 1045-1048 (2010).</li><li>Encode Project Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+user's+guide+to+the+encyclopedia+of+DNA+elements+(ENCODE).">A user's guide to the encyclopedia of DNA elements (ENCODE).</a> PLoS Biol. 9, e1001046 (2011).</li><li>Asp, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+remodeling+of+the+epigenetic+landscape+during+myogenic+differentiation.">Genome-wide remodeling of the epigenetic landscape during myogenic differentiation.</a> Proc Natl Acad Sci U S A. 108, E149-E158 (2011).</li><li>Hoppe, P. S., Coutu, D. L., Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+technologies+sharpen+up+mammalian+stem+cell+research.">Single-cell technologies sharpen up mammalian stem cell research.</a> Nat Cell Biol. 16, 919-927 (2014).</li><li>Avital, G., Hashimshony, T., Yanai, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+is+believing:+new+methods+for+in+situ+single-cell+transcriptomics.">Seeing is believing: new methods for in situ single-cell transcriptomics.</a> Genome Biol. 15, 110 (2014).</li><li>Navin, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+genomics:+one+cell+at+a+time.">Cancer genomics: one cell at a time.</a> Genome Biol. 15, 452 (2014).</li><li>Saliba, A. E., Westermann, A. J., Gorski, S. A., Vogel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+RNA-seq:+advances+and+future+challenges.">Single-cell RNA-seq: advances and future challenges.</a> Nucleic Acids Res. 42, 8845-8860 (2014).</li><li>Nagano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+Hi-C+reveals+cell-to-cell+variability+in+chromosome+structure.">Single-cell Hi-C reveals cell-to-cell variability in chromosome structure.</a> Nature. 502, 59-64 (2013).</li><li>Kind, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+dynamics+of+genome-nuclear+lamina+interactions.">Single-cell dynamics of genome-nuclear lamina interactions.</a> Cell. 153, 178-192 (2013).</li><li>Southall, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-type-specific+profiling+of+gene+expression+and+chromatin+binding+without+cell+isolation:+assaying+RNA+Pol+II+occupancy+in+neural+stem+cells.">Cell-type-specific profiling of gene expression and chromatin binding without cell isolation: assaying RNA Pol II occupancy in neural stem cells.</a> Dev Cell. 26, 101-112 (2013).</li></ol>

The authors have nothing to disclose.

Whether Dam-tagged proteins retain the functions of endogenous proteins should be examined before a DamID-seq experiment. The subcellular localization of Dam-tagged nuclear envelope proteins should always be determined and compared with that of the endogenous proteins. For studying transcription factors, it is suggested to examine whether the Dam-fusion protein can rescue the functions of the endogenous protein in regulating gene expression. This functional test can be performed in organisms in which knockout mutants of endogenous DNA-binding proteins are available. Because advances in genome engineering have potentially allowed knocking out any endogenous gene of interest, functions of Dam-tagged DNA-binding proteins can be examined in cultured mammalian cells. 
The critical step in this protocol is to successfully fragment the DpnII-digested DamID PCR products to around 200 bp. This step is designed to render the amplified adenine-methylated fragments to a narrow size range for sequencing and to randomize the starting nucleotides of the DNA fragments in a sequencing library. Inefficient fragmentation will leave the majority of the DNA fragments starting with GATC (the 5'-overhang from the second DpnII digestion), and will result in a much lower performance and yield or even a failure in Illumina sequencing. Other DNA fragmentation methods may be used as an alternative approach. 
The resolution of DamID (and DamID-seq described here) is limited by the frequency of GATCs in the genome to be studied. Moreover, even with high throughput sequencing, the genomic localizations of a DNA-binding protein can only be mapped within two consecutive GATCs rather than to the actual DNA-binding sites. 
Despite its limitation, the DamID assay has important advantages. Because DamID does not require highly-specific antibodies, it can be used to detect a subset of nuclear proteins that could be difficult to assay by ChIP (such as the nuclear envelope proteins). To study how these proteins regulate genome functions, it is important to integrate and cross-analyze their genome-wide localization data with the current epigenomic mapping data (such as data from the ENCODE and NIH Roadmap Epigenomics Projects 30,31). The DamID-seq approach provides both higher resolution and higher sensitivity than DamID by microarray and enables detecting differential NL-associations within gene structures 10. A combinatorial analysis of DamID-seq data, ChIP-seq data 32 and gene expression data has identified a class of NL-associated genes with distinct epigenetic and transcriptional features (data not published). 
Another advantage of DamID is that it only requires a small number of cells. In recent years, there has been an explosion in single cell analysis of gene regulation 33,34. Although genome sequence 35, genome-wide gene expression 36 and chromatin conformation 37 can be assayed in a single cell, there has not been an available approach for detecting protein-DNA interactions genome-wide in a single cell. DamID-seq is a highly promising approach for this goal, and may complement the single cell imaging approach in detecting the dynamics of genome-NL interactions 38. One complication is that because the Dam-fusion protein is expressed at a much lower level than the endogenous protein in the DamID assay, it is possible that the Dam-fusion protein may only occupy a subset of genomic binding sites as compared to the endogenous protein. 
DamID assay has mostly been used in cultured animal cells to detect protein-DNA interactions. Notably, developmental biologists have applied this assay in detecting protein-DNA interactions in specific cell types in vivo. For example, Dam-tagged RNA polymerase II was expressed specifically in Drosophila neural stem cells to detect their genome-wide occupancy without cell isolation 39. DamID-seq will be highly useful to study the genome-wide localizations of nuclear envelope proteins, transcription factors and chromatin regulators during development in animal models.

脱氧核糖核酸腺嘌呤甲基识别（DamID）1,2是这样一种方法来检测体内蛋白-DNA相互作用，是一种可供选择的方法来染色质免疫沉淀（ChIP）3。它采用的是相对低的细胞的量，并且不需要化学交联蛋白与DNA或高度特异性的抗体。后者是特别有用当靶蛋白是松散或间接地与DNA相关联。 DamID已成功地用于绘制的多种蛋白质，包括核包膜蛋白4-10，相关联的染色质蛋白11-13染色质修饰酶14，转录因子和辅因子15-18和RNAi机械19的结合位点。该方法适用于多种生物，包括S.酵母 13，S。酵母 7，C。线虫 9,17，D。黑 5,11,18，20，A。拟南芥 21,22以及小鼠和人的细胞系6,8,10,23,24。 所述DamID测定的发展是基于在缺乏内源性腺嘌呤甲基化2的真核细胞腺嘌呤甲基化的DNA片段的特异性检测。表达的融合蛋白，包括利息和 E的DNA结合蛋白的大肠杆菌DNA腺嘌呤甲基（水坝），可以甲基化腺嘌呤碱中GATC序列是在空间接近性（最显著内1 kb和多达大约5kb的）的蛋白质的基因组中的2的结合位点。修饰的DNA片段可以特异性地扩增和杂交到微阵列检测感兴趣1,25,26的蛋白质的基因组结合位点。这个原始DamID方法是由微阵列的可用性和预定探针的密度的限制。因此，我们集成了高通量测序到DamID 10和指定的方法DamID-起。读取从DamID-SEQ产生大量的短使蛋白质-DNA相互作用的全基因组的精确定位。我们发现，DamID-SEQ提供比DamID更高的分辨率和更宽的动态范围芯片用于研究基因组核纤层（NL）协会10。这种改进的方法允许基因结构 10内探测NL协会和便于与其它高通量测序数据，如芯片起和RNA-SEQ比较。 这里描述的DamID-SEQ协议最初开发用于映射基因组-NL协会10。我们通过圈养小鼠或人类核纤层蛋白B1至E.产生的融合蛋白大肠杆菌DNA甲基腺嘌呤和测试协议中3T3小鼠胚胎成纤维细胞，C2C12小鼠成肌细胞10和IMR90人胚肺成纤维细胞（数据未公布）。在这个协议中，我们开始用Constructing载体和表达坝-拴系融合蛋白由慢病毒感染的哺乳动物细胞24。接着，我们描述放大腺嘌呤甲基化的DNA片段，并准备测序文库，应该适用于其它生物体的详细方案。

<table><tbody><tr><td>ViraPower Lentiviral Expression Systems</td><td>Life Technologies</td><td>K4950-00, K4960-00, K4970-00, K4975-00, K4980-00, K4985-00, K4990-00, K367-20, K370-20, and K371-20</td><td></td></tr><tr><td>Gateway BP Clonase II Enzyme Mix</td><td>Life Technologies</td><td>11789-020</td><td></td></tr><tr><td>Gateway LR Clonase II Enzyme Mix</td><td>Life Technologies</td><td>11791-020</td><td></td></tr><tr><td>DNeasy Blood &amp;amp; Tissue Kit (250)</td><td>QIAGEN</td><td>69506 or 69504&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Gateway pDONR 201</td><td>Life Technologies</td><td>11798-014</td><td></td></tr><tr><td>293T cells</td><td>American Type Culture Collection</td><td>CRL-11268</td><td></td></tr><tr><td>&lt;a href=&quot;http://www.lifetechnologies.com/order/catalog/product/25300054&quot;&gt;Trypsin-EDTA (0.05%), phenol red&lt;/a&gt;</td><td>Life Technologies</td><td>25300-054</td><td></td></tr><tr><td>DMEM, high glucose, pyruvate</td><td>Life Technologies</td><td>11995-065</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma</td><td>F4135</td><td></td></tr><tr><td>Tris</td><td></td><td></td><td>brand not critical</td></tr><tr><td>EDTA</td><td></td><td></td><td>brand not critical</td></tr><tr><td>200 Proof EtOH</td><td></td><td></td><td>brand not critical</td></tr><tr><td>Isopropanol</td><td></td><td></td><td>brand not critical</td></tr><tr><td>Sodium Acetate</td><td></td><td></td><td>brand not critical</td></tr><tr><td>DpnI</td><td>New England Biolabs</td><td>R0176</td><td>supplied with buffer</td></tr><tr><td>DamID adaptors &quot;AdRt&quot; and &quot;AdRb&quot;</td><td>Integrated DNA Technologies</td><td></td><td>sequences available in ref. 24; no phosphorylation of the 5&#039; or 3&#039; end to prevent self-ligation.</td></tr><tr><td>T4 DNA Ligase</td><td>Roche Life Science</td><td>10481220001</td><td>supplied with buffer</td></tr><tr><td>DpnII</td><td>New England Biolabs</td><td>R0543</td><td>supplied with buffer</td></tr><tr><td>DamID PCR primer &quot;AdR_PCR&quot;</td><td>Integrated DNA Technologies</td><td></td><td>sequences available in ref. 24</td></tr><tr><td>Deoxynucleotide (dNTP) Solution Set</td><td>New England Biolabs</td><td>N0446</td><td>100 mM each of dATP, dCTP, dGTP and dTTP</td></tr><tr><td>Advantage 2&amp;nbsp; Polymerase Mix</td><td>Clontech</td><td>639201</td><td>supplied with buffer</td></tr><tr><td>1Kb Plus DNA Ladder</td><td>Life Technologies</td><td>10787018</td><td>1.0 &amp;micro;g/&amp;micro;l</td></tr><tr><td>QIAquick PCR Purification Kit</td><td>QIAGEN</td><td>28104 or 28106</td><td></td></tr><tr><td>MinElute PCR Purification Kit</td><td>QIAGEN</td><td>28004 or 28006</td><td>for an elution volume of less than 30 &amp;micro;l</td></tr><tr><td>SPRI beads / Agencourt AMPure XP</td><td>Beckman Coulter</td><td>A63880</td><td>apply extra mixing and more elution time if less than 40 &amp;micro;l elution buffer is used</td></tr><tr><td>Buffer EB</td><td>QIAGEN</td><td>19086</td><td></td></tr><tr><td>NEBNext dsDNA Fragmentase</td><td>New England Biolabs</td><td>M0348</td><td>supplied with buffer</td></tr><tr><td>T4 DNA Ligase Reaction Buffer</td><td>New England Biolabs</td><td>B0202</td><td></td></tr><tr><td>T4 DNA Polymerase</td><td>New England Biolabs</td><td>M0203</td><td></td></tr><tr><td>DNA Polymerase I, Large (Klenow) Fragment</td><td>New England Biolabs</td><td>M0210</td><td></td></tr><tr><td>T4 Polynuleotide Kinase</td><td>New England Biolabs</td><td>M0201</td><td></td></tr><tr><td>Klenow Fragment (3&amp;rsquo; &amp;#8594; 5&amp;rsquo; exo-)</td><td>New England Biolabs</td><td>M0212</td><td>supplied with buffer</td></tr><tr><td>sequencing adaptors</td><td>Integrated DNA Technologies</td><td></td><td>sequences available in ref. 28</td></tr><tr><td>Quick Ligation Kit</td><td>New England Biolabs</td><td>M2200</td><td>used in 3.4.3; supplied with Quick Ligation Reaction Buffer and Quick T4 DNA Ligase</td></tr><tr><td>sequencing primer 1 and 2</td><td>Integrated DNA Technologies</td><td></td><td>sequences available in ref. 28</td></tr><tr><td>KAPA HiFi PCR Kit</td><td>Kapa Biosystems</td><td>KK2101 or KK2102</td><td>supplied with KAPA HiFi DNA Polymerase, 5x KAPA HiFi Fidelity Buffer and 10 mM dNTP mix</td></tr><tr><td>agarose</td><td>Sigma Aldrich</td><td>A4679</td><td></td></tr><tr><td>ethidium bromide</td><td>Sigma Aldrich</td><td>E1510-10ML</td><td>10 mg/ml</td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>QIAGEN</td><td>28704 or 28706</td><td></td></tr><tr><td>iTaq Universal SYBR Green Supermix</td><td>Bio-Rad Laboratories</td><td>1725121 or 1725122</td><td></td></tr><tr><td>Spectrophotometer</td><td></td><td></td><td>brand not critical</td></tr><tr><td>0.45 &amp;#956;m PVDF Filter</td><td></td><td></td><td>brand not critical</td></tr><tr><td>25 ml Seringe</td><td></td><td></td><td>brand not critical</td></tr><tr><td>10 cm Tissue Culture Plates</td><td></td><td></td><td>brand not critical</td></tr><tr><td>6-well Tissue Culture Plates</td><td></td><td></td><td>brand not critical</td></tr><tr><td>S1000 Thermal Cycler</td><td>Bio-Rad Laboratories</td><td></td><td></td></tr><tr><td>C1000 Touch Thermal Cycler</td><td>Bio-Rad Laboratories</td><td></td><td>for qPCR</td></tr><tr><td>Vortex Mixer</td><td></td><td></td><td>brand not critical</td></tr><tr><td>Dry Block Heater or Thermomixer</td><td></td><td></td><td>brand not critical</td></tr><tr><td>Microcentrifuge</td><td></td><td></td><td>brand not critical</td></tr><tr><td>Gel electrophoresis system with power supply</td><td></td><td></td><td>brand not critical</td></tr><tr><td>Magnet stand</td><td></td><td></td><td>for purification of DNA with SPRI beads; should hold 1.5-2 ml tubes; brand not critical</td></tr><tr><td>UV transilluminator</td><td></td><td></td><td>brand not critical</td></tr><tr><td>E-gel electrophoresis system</td><td>Life Technologies</td><td>G6400, G6500, G6512ST</td><td></td></tr></tbody></table>

damid-seq: genome-wide mapping of protein-dna interactions by high throughput sequencing of adenine-methylated dna fragments

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

大坝-V5-LmnB1融合蛋白被确认已共定位与内源拉明乙蛋白通过免疫荧光染色（ 图1）。 腺嘌呤甲基化DNA片段的成功的PCR扩增为DamID-SEQ的关键步骤。实验样品应扩增的0.2污迹- 2 kb的，而阴性对照（无DPNI，没有连接酶或不含PCR模板）应导致无或明显较少的扩增（ 图2）。 甲基化DNA片段是在0.2至2kb的范围内，而所希望的插入大小为一个NGS文库是从200至300bp。因此，有必要将其分解的甲基PCR产物插入合适的尺寸范围。尽管如此，它被认为是不切实际的，同时破坏较大的DNA片段，下降到合适的大小S和保持广大小DNA片段完整的单个碎片的持续时间。因此，时间过程进行实验，以确定所需的片段1微克的DNA，以中心在200bp的（图3）涂抹标本的最小时间（T 0.2kb）。然后6持续时间在等额递增5分钟和T 0.2kb实际碎片之间进行选择。双链DNA Fragmentase的酶活性可从批次不同而不同批次，并可能随时间降低，因此建议以重复对新一批Fragmentase的或储存一段时间后这一步。 所需的插入大小为200至300碱基对对应于300和400碱基对（包括121 bp的测序接头）之间的DNA片段上琼脂糖凝胶。在此范围内三个薄片从每个实验样品切下，以缩小的文库的尺寸范围和增加的可能性的获得至少一个合格的测序文库（图4）。 5微升每个扩增的​​DNA文库的等分试样在琼脂糖凝胶上分析，以确定哪些文库可以有资格进行测序。 如图5A所示，大小相同的切凝胶切片的明确单一带应该在琼脂糖凝胶（步骤3.7.4）可见。接着，选定的库进行了检查用生物分析仪（图5B）之前，以确定要测序的确切大小范围和浓度。如果需要的话，扩增的DNA文库可以直接检查由一个生物分析仪没有凝胶分析。当多个库的质量是好的，建议进行测序相似大小的文库范围为一对实验（细胞表达坝-V5-POI）和对照的（细胞表达V5-坝）样品。 短读取所产生的测序系统最初映射回相应的基因组中。独特对准读取，然后传递给后续分析。一个管道来处理快速读取，构建基因组-NL互动地图和分析基因-NL协会进行了详细的前期工作10描述。代表性的结果示于图6。 <img alt="图1" src="/files/ftp_upload/53620/53620fig1.jpg" /> 图1.通过免疫荧光染色验证融合蛋白的亚核本地化。IMR90人肺成纤维细胞瞬时转染表达坝-V5-LmnB1的质粒和由抗拉明乙（ 甲 ，红色）和抗V5（B进行染色，绿色）。 （三）合并A和B中的图像<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53620/53620fig1large.jpg" target="_blank">，请点击这里查看T的放大版</a>他的身影。 <img alt="图2" src="/files/ftp_upload/53620/53620fig2.jpg" /> 图2.使用PCR扩增腺嘌呤甲基化的DNA片段。5微升从每个PCR反应的等分试样上1％琼脂糖凝胶上分析。涂片范围从0.2到2 KB，从每个实验样品扩增，但没有扩增的阴性对照观察（无DPNI步骤2.1，步骤2.2，或者没有PCR模板没有连接酶）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53620/53620fig2large.jpg" target="_blank">请点击此处查看大图这个数字。</a> <img alt="图3" src="/files/ftp_upload/53620/53620fig3.jpg" /> 图3.确定最佳碎片的持续时间。纯化的甲基PCR产物受到碎片为持续时间从5至55分钟，在10分钟的增量（未消化的DNA<html> ，标示为"0分"）。 1微克的DNA梯和0.5微克每片段化DNA样品取在琼脂糖凝胶上进行分析。在最短的时间内的大多数的DNA涂片消化约200bp的测定为约35分钟，因此6 5至35分钟（5和35分钟在内）均匀间隔开的时间的持续时间被选择来执行实际的碎片。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53620/53620fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53620/53620fig4.jpg" /> 图4.大小选择的DNA文库。坝-V5-LmnB1和V5-大坝DNA样品来自步骤3.6中在2％琼脂糖凝胶上运行，并且三个相等大小的凝胶切片（L，M和H对应于低，中和尺寸高）的300和400碱基对之间切下如由黄线。<html> OM /文件/ ftp_upload / 53620 / 53620fig4large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图5" src="/files/ftp_upload/53620/53620fig5.jpg" /> 在琼脂糖凝胶上分析图5扩增的DNA文库进行高通量测序。（A）的扩增的DNA文库。 PCR模板是从图4所示的凝胶片法（ 二）V5-坝（L）样本与大坝-V5-LmnB1（L）样品，强调了（A）表示，生物分析仪的结果。这两个库有类似窄的尺寸范围，因此合格的高通量测序。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53620/53620fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> JPG"/&gt; 在UCSC基因组浏览器。小鼠染色体1 显示图6. NGS数据被示出为示例。序列数据是从小鼠成肌细胞C2C12 10产生 。曲目"MB.LmnB1.w2k"和"MB.Dam.w2k"，对应于从细胞中分别表达大坝-V5-LmnB1和V5-水坝的数据，积归读密度（每千个碱基每百万唯一映射读取或RPKM读）的非重叠连续2 KB的窗户沿着染色体。轨道"MB.log2FC.w2k"地块基因组NL协会， 即 LOG2 LmnB1的RPKM比例过大坝，在2 KB的分辨率。轨道"MB.sLADs"描绘测序基薄层相关域（sLADs，有LmnB1的更高的读取密度过大坝具有统计学意义，即基因组区域）黑色，非sLADs在白灰色和不确定的地区。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53620/53620fig6large.jpg" target="_blank">请点击此处downloa</a>d此文件。

Watch this Scientific Journal Video about DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments at JoVE.com

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

我们描述这里的一个检测通过结合DNA腺嘌呤甲基识别（DamID），以高通量测序（DamID-SEQ）。这种改进的方法提供了更高的分辨率和更宽的动态范围，并允许在分析与其他高通量测序数据如芯片起，RNA测序等一起DamID-SEQ数据

DamID-SEQ:蛋白质-DNA相互作用的全基因组映射腺嘌呤甲基化的DNA片段通过高通量测序

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

damid-seq-genome-wide-mapping-protein-dna-interactions-high

Universidad San Sebastian

Research

JoVE Journal

Biology

19.4K 次观看.  Yale University School of Medicine. 我们描述这里的一个检测通过结合DNA腺嘌呤甲基识别（DamID），以高通量测序（DamID-SEQ）。这种改进的方法提供了更高的分辨率和更宽的动态范围，并允许在分析与其他高通量测序数据如芯片起，RNA测序等一起DamID-SEQ数据

视频: DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

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 ...

Informatic Analysis of Sequence Data from Batch Yeast 2-Hybrid Screens

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing high-throughput short-read DNA sequencing. The resulting sequence datasets can not only track what genes in a population that are enriched during selection for positive yeast 2-hybrid interactions, but also give detailed information about the relevant subdomains of proteins sufficient for interaction. Here, we describe a full suite of stand-alone software programs that allow non-experts to perform all the bioinformatics and statistical steps to process and analyze DNA sequence fastq files from a batch yeast 2-hybrid assay. The processing steps covered by these software include: 1) mapping and counting sequence reads corresponding to each candidate protein encoded within a yeast 2-hybrid prey library; 2) a statistical analysis program that evaluates the enrichment profiles; and 3) tools to examine the translational frame and position within the coding region of each enriched plasmid that encodes the interacting proteins of interest.

Deep sequencing of yeast populations selected for positive yeast 2-hybrid interactions potentially yields a wealth of information about interacting partner proteins. Here, we describe the operation of specific bioinformatics tools and customized updated software to analyze sequence data from such screens.

间歇酵母 2-混合筛序列数据的信息化分析

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing ...

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 ...

Mapping Mammalian 3D Genome Interactions with Micro-C-XL

Three-dimensional (3D) chromosome organization is a major factor in genome regulation and cell-type specification. For example, cis-regulatory elements, known as enhancers, are thought to regulate the activity of distal promoters via interaction in 3D space. Genome-wide chromosome conformation capture (3C)-technologies, such as Hi-C, have transformed our understanding of how genomes are organized in cells. The current understanding of 3D genome organization is limited by the resolution with which the topological organization of chromosomes in 3D space can be resolved. Micro-C-XL measures chromosome folding with resolution at the level of the nucleosome, the basic unit of chromatin, by utilizing micrococcal nuclease (MNase) to fragment genomes during the chromosome conformation capture protocol. This results in an improved signal-to-noise ratio in the measurements, thus facilitating the better detection of insulation sites and chromosome loops compared to other genome-wide 3D technologies. A visually supported, detailed, step-by-step protocol for preparing high-quality Micro-C-XL samples from mammalian cells is presented in this article.

A protocol for mapping the three-dimensional genome organization with nucleosome resolution using the genome-wide chromosome conformation capture method Micro-C-XL is presented here.

使用 Micro-C-XL 绘制哺乳动物 3D 基因组相互作用图

Three-dimensional (3D) chromosome organization is a major factor in genome regulation and cell-type specification. For example, cis-regulatory elements, ...

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

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation1. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs2.
Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP)3-5. These approaches were prone to identifying indirect or non-physiological interactions6. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced7,8. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq)9,10. Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking11,12.
Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide13. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1)14. Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation14.

The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.

iCLIP - 全转录组，蛋白质与RNA相互作用的映射与个别核苷酸分辨率

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional ...

生物学

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 ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

探讨高密度功能蛋白微阵列来检测蛋白质 - 蛋白质相互作用

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

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).

DamID-SEQ:蛋白质-DNA相互作用的全基因组映射腺嘌呤甲基化的DNA片段通过高通量测序

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与ChEC-seq的蛋白质-DNA相互作用的全基因组映射