作者感谢ULE，勒斯科姆和祖潘实验室的所有成员，讨论和实验援助。我们感谢詹姆斯哈德菲尔德和尼克马修斯高通量测序。我们想指出，iCLIP方法这里描述的几个步骤股份与原始剪辑协议，柯克詹森和居开发实验室的罗伯特达内尔。这项工作是支持由欧洲研究理事会授予206726剪辑柱和一个长期的人类前沿科学计划奖学金JK

1。 UV交联的组织培养细胞<ol><li>取出介质，并加入6毫升冰冷的PBS细胞生长在一个10厘米的板（足够的三个实验）。 </li><li>取下盖板，将在冰上。 150兆焦耳/厘米2在254 nm，照射一次。 </li><li>收获的细胞与细胞起苗刮。 </li><li>将2毫升细胞悬液，每3微管。最高时速为10秒旋转4 ° C至沉淀细胞，然后去除上清液。 </li><li>管理单元冻结在-80 ° C，直到使用干冰和存储单元颗粒。 </li></ol> 2。珠准备<ol><li>加入100μL蛋白一个磁珠每到一个新的微管实验（DYNAL 100.02）（使用鼠标或山羊抗体蛋白G磁珠）。 </li><li>洗珠裂解液（50毫米的Tris - HCl，pH值7.4; 100毫米氯化钠，1％NP - 40，0.1％SDS，0.5％去氧胆酸钠，1 / 100的蛋白酶抑制剂的鸡尾酒第三，Calbiochem）的2倍。 </li><li>在2-10微克抗体100μL裂解液重悬珠。 </li><li>在室温为30-60分钟旋转管。 </li><li> 900μL裂解液洗3倍，并准备继续执行步骤4.1离开，直到在最后一次洗涤。 </li></ol> 3。细胞裂解和RNA的消化部分<ol><li> 1毫升裂解液转移到1.5 ml的微管，重悬细胞沉淀。 </li><li>我核糖核酸（Ambion公司，AM2295）准备一个1 / 500稀释。添加10μL核糖核酸我稀释以及2μL涡轮的DNA酶的细胞裂解液（核糖核酸我1 / 500稀释低RNase的1 / 50稀释用于库准备; [高核糖核酸酶抗体的特异性是必要的控制） 。 </li><li>孵育整整3分钟，在37 ° C在1100 rpm时的晃动，样品。立即传送到冰。 </li><li>旋转4 ° C和20分钟的22000克，以明确的裂解。仔细收集上清（留约50μL裂解液沉淀）。 </li></ol> 4。免疫沉淀<ol><li>删除从珠（步骤2.5）缓冲液洗，再加入细胞裂解液（步骤3.4）。 </li><li>旋转2 h后的样品在4 ° C。 </li><li>弃上清，洗珠900μL高盐缓冲液（50毫米的Tris -盐酸，pH值7.4; 1 M氯化钠; 1毫米EDTA，1％NP - 40，0.1％SDS，0.5％去氧胆酸钠）2X。 </li><li>洗2倍，而与900μL洗涤缓冲液（20毫米的Tris - HCl，pH值7.4; 10毫米MgCl 2的 0.2％Tween - 20的）。 </li></ol> 5。去磷酸化的RNA的3&#39;ends <ol><li>弃上清，重悬在20μLPNK混合珠（15μl水; 4μL5倍PNK pH值6.5缓冲液[350mMTris盐酸，pH值6.5; 50mMMgCl 2 25mMdithiothreitol]; 0.5μLPNK酶0.5μLRNasin [Promega公司]） 。 </li><li> 20分钟在37 ° C。 </li><li>加入500μl洗涤缓冲液，并用高盐缓冲液1X。 </li><li>洗涤缓冲液洗2倍。 </li></ol> 6。链接器结扎RNA 3&#39;末端<ol><li>小心取出上清液，重悬在20μL结扎混合珠（9μl水; 4μL4X连接缓冲液[200 mMTris -盐酸;40米MM GCL 2; 40毫米二硫苏糖醇]; 1μLRNA连接酶0.5μL，RNasin [NEB]; Promega公司] 1.5μL预adenylated连接器L3 [20微米; 4μLPEG400 [81170，Sigma公司）。 </li><li>过夜孵育在16 ° C。 </li><li>加入500μl洗涤缓冲液，然后用1毫升高盐缓冲的2倍。 </li><li>洗用1 ml缓冲液洗2倍和1毫升洗第二次离开。 </li></ol> 7。 RNA的5&#39;末端标记<ol><li>取出上清液，重悬在8μL热PNK混合珠（0.4μLPNK [NEB]; 0.8μL32个P -γ- ATP的0.8μL10X PNK缓冲[NEB]; 6μl水）。 </li><li> 5分钟在37 ° C。 </li><li>取出热PNK组合和20μL1X Nupage样缓冲液（Invitrogen公司）重悬珠。 </li><li>在thermomixer孵育70 ° C时为10分钟。 </li><li>立即将一块磁铁沉淀空珠和负载上清液凝胶（见第8步）。 </li></ol> 8。 SDS - PAGE和膜内转印<ol><li>负载的样品在4-12％NuPAGE双三凝胶（Invitrogen公司）根据制造商的指示。使用1X MOPS运行缓冲液（Invitrogen）的0.5升。还可以加载5μL预染蛋白大小的标记（例如页统治者加，Fermentas公司，SM1811）。 </li><li>运行50分钟的凝胶在180 V。 </li><li>取出凝胶前和丢弃的固体废物（含免费放射性ATP的）。 </li><li>蛋白- RNA复合物，从凝胶转移到硝酸纤维素膜，使用NOVEX调湿装置，根据制造商的说明（Invitrogen公司，转让1小时在30 V）。 </li><li>转让后，PBS缓冲液冲洗膜，然后在保鲜膜包装，并到富士胶片暴露于-80 ° C（地方一个荧光贴纸膜后ALIGN T他的膜和膜30分钟，1小时，在夜间执行曝光）。 </li></ol> 9。 RNA分离<ol><li>从低核糖核酸作为面膜的步骤8.5中使用的放射自显影的实验分离蛋白- RNA复合物。这片膜切成若干小片，放入1.5 ml的微管。 </li><li>添加200μLPK缓冲区（100毫米的Tris - HCl pH 7.4的50 mM氯化钠10毫米EDTA）和10μL蛋白酶K（罗氏，03115828001）膜件。孵育20分钟1100转的摇晃，在37 ° C。 </li><li> PKurea缓冲区（100毫米的Tris - HCl pH 7.4的50 mM氯化钠10毫米EDTA; 7 M尿素）加入200μL和20分钟在37 ° C。 </li><li>收集解决方案，并添加400μLRNA的酚/氯仿（Ambion公司，9722）2毫升锁相凝胶重型管（713-2536，VWR）。 </li><li>孵育5分钟在30 ° C，在1100 rpm时的晃动。 13000 RPM在室温5分钟旋转分离的阶段。 </li><li>转移到一个新的管水层（要小心，不要触摸移液器凝胶）。添加0.5μLglycoblue（Ambion公司，9510）和40μL3 M醋酸钠pH值5.5和混合。然后加入1 ml 100％的乙醇，再次组合，在夜间沉淀于-20 ° C </li></ol> 10。反转录<ol><li> 20分钟转速15,000 RPM和4 ° C。去除上清液，并用0.5毫升80％乙醇沉淀。 </li><li> 7.25μLRNA /引物组合（6.25μl水0.5μLRclip底漆[0.5pmol/μl]; 0.5μLdNTP混合液[10毫米），重悬沉淀。对于每个实验或复制，使用不同Rclip的引物，包含个人的条码序列（见14）。 </li><li>孵育5分钟，在70℃前冷却至25 ° C。 </li><li>添加2.75μLRT组合（2μL的5倍RT缓冲; 0.5μL0.1M数码地面电视服务; 0.25μL标III逆转录Invitrogen公司]）。 </li><li>孵育5分钟在25℃，20分钟，42℃，40分钟，在50 ° C和5分钟，在80℃前冷却至4 ° C。 </li><li>加入90μLTE缓冲液，0.5μLglycoblue，10μL醋酸钠pH值5.5和混合。然后加入250μL100％的乙醇，混合和过夜沉淀于-20 ° C </li></ol> 11。凝胶纯化的cDNA <ol><li>降速和洗的样本（见10.1），然后重新悬浮颗粒在水6μL。 </li><li>加入6μL的2倍TBE -尿素样缓冲液（Invitrogen公司）。加热样品至80 ° C为直接装载前3分钟。 </li><li>装入预制6％TBE -尿素凝胶（Invitrogen）的样品和制造商的180 V 40分钟运行。此外，负载低分子量标记，为后续的切削（见下文）。 </li><li>削减120-200新台币（高），85-120 NT（中）和70-85 NT（低）三个波段。使用theupper染料，塑料凝胶支持的标志，引导切除术（见图3）。请注意，Rclip底漆和三层序列合计占52 NT的剪辑顺序。 </li><li>加入400μLTE和粉碎成小块，用1毫升注射器的柱塞的凝胶片。孵育2小时在1100 RPM晃动在37 ° C。 </li><li>将两个1厘米的玻璃预过滤器进入一个中光学SpinX列（康宁公司，8161）（滤纸，1823010）。液体样品的部分转移到列。自旋为1分钟到1.5 ml管13000 RPM。 </li><li>添加0.5μLglycoblue和40μL醋酸钠的pH值5.5，然后混合样本。添加1毫升100％的乙醇，重新组合和过夜沉淀在-20 ° C。 </li></ol> 12。 cDNA的5&#39;端引物的结扎<ol><li>降速和洗的样本（见10.1），然后重新悬浮颗粒在8μL结扎组合（6.5μl水，0.8μL10倍CircLigase缓冲区第二; 0.4μL50毫米MnCl 2; 0.3μL; Circligase II [震中]），并培育在60 ° C 1小时</li><li>加入30μl的寡核苷酸退火组合（26μl水; 3μLFastDigest缓冲[Fermentas公司; 1μLcut_oligo [10μM]）。孵育1分钟，在95 ° C。然后下降的温度每20秒1 ° C到25 ° C是达到了。 </li><li> 2μLBamHI位（快速Fermentas公司）在37和30分钟的孵化° C。 </li><li>加入50μLTE和0.5μLglycoblue和混合。添加10μL醋酸钠pH值5.5和混合，再加入250μL100％的乙醇。再次在夜间混合和沉淀于-20 ° C </li></ol> 13。 PCR扩增<ol><li>降速和洗的样本（见10.1），然后在19μl水重悬沉淀。 </li><li>准备PCR混合物（19μL的cDNA; 1μL引物组合P5/P3公司Solexa，每10μM; 20μLAccuprime Supermix 1酶[Invitrogen公司]）。 </li><li>运行下面的PCR程序：94℃15秒，65℃30秒，30秒，68 ° C] 25-35个循环，68 ° C为3分，4个94 ° C，2分钟， ° C直到永远。 </li><li>混合8μLPCR产物与2μL预制6％TBE凝胶上装载的5倍TBE缓冲和负载（Invitrogen）。 Sybrgreen我（Invitrogen）的凝胶染色，用凝胶成像仪分析。 </li><li>在Rclip引物的条形码允许复用不同的样品的高通量测序之前提交。提交15μL测序库和存储休息。 </li></ol> 14。链接程序和引物序列前adenylated 3&#39;连接器的DNA： 我们为了从IDT的DNA适配器，然后分装的20μM。] <img src="/files/ftp_upload/2638/2638fig0.jpg" alt="的DNA" /> 15。代表性的成果： 测序iCLIP库之前，实验的成功，可以监视两个步骤：膜内转印后的蛋白质- RNA复合物（步骤8.5）和PCR产物的凝胶图像（13.4步）放射自显影。在低核糖核酸样本的放射自显影，弥漫性放射性应被视为上述分子量的蛋白质（图2，样品4）。对于高核糖核酸样本，这种放射性是接近的蛋白质的分子量（图2，样品3）集中。如果没有抗体是免疫，应检测无信号（图2，样品1和2）。特异性免疫的进一步重要的控制，要么省略紫外线照射，或使用不表达的细胞蛋白质的利息14。 的PCR产物（13.4步）的凝胶图像显示大小的范围内，对应纯化步骤11.4（图4，泳道4-6）cDNA的部分（高，中，低）。请注意，PCR引物P3Solexa和P5Solexa，引入一个额外的76 NT的cDNA的大小。如果没有抗体的免疫过程中使用，没有相应的PCR产物（图4，泳道1-3）应检测。引物二聚体的产品可以出现在约140 NT。 对于代表的高通量的测序和随后生物信息学分析结果，看到14。 <img src="/files/ftp_upload/2638/2638fig1.jpg" alt="图1" /> 图1。iCLIP协议的示意图。蛋白质- RNA复合物共价交联用紫外线照射（步骤1）体内。感兴趣的蛋白质纯化与结合的RNA（步骤2-5）。为了让序列特异性逆转录吸，一种RNA适配器是RNA的3&#39;端结扎，而放射性标记的5&#39;端（步骤6和7）。交联蛋白- RNA复合物是免费的RNA纯化，用SDS - PAGE和膜内转印（步骤8）。 RNA是从膜回收，通过消化的蛋白质与蛋白酶K留在交叉链接核苷酸（步骤9）多肽其余。反转录（RT）在余下的多肽截断，并介绍了两种裂解适配器地区和条形码序列（10步）。大小选择删除前函证免费RT引物。下面的线性生成合适的模板进行PCR扩增（步骤11-15）。最后，高通量测序生成读取条形码序列会立即被最后的cDNA核苷酸（16步）。由于这核苷酸位于一个位置的交联核苷酸上游，结合部位，可以推导出高分辨率。 <img src="/files/ftp_upload/2638/2638fig2.jpg" alt="图2" /> 图2交联的核蛋白的C - RNA复合物的放射自显影使用变性凝胶电泳和膜转移。 （α核蛋白核蛋白的C - RNA复合物从细胞提取核蛋白彗星对使用一种抗体免疫纯化，样品3和4）。 RNA部分被消化，使用低（+）或高（+）浓度的RNase。可以观察到向上移位大小的蛋白质（40 kDa）的配合物（样品4）。这种转变是不太明显，当高浓度的RNase（样品3）。放射性信号消失时没有抗体用于免疫沉淀（样品1和2）。 <img src="/files/ftp_upload/2638/2638fig3.jpg" alt="图3" /> 图3。示意图6％TBE -尿素凝胶（Invitrogen公司），以指导切除iCLIP cDNA产物。凝胶是运行在180 V，导致重复的迁移模式的cDNA和染料在凝胶（光与暗蓝色）为40分钟。使用刀片切割（红线）高（H），中（M），低（L）cDNA的分数。开始在中间的淡蓝色染料，并立即上述商标切割塑料胶盒。划分中，低分数和修剪约1厘米以上的淡蓝色染料的高分数。使用的口袋和染料的指导下的垂直切割，分开不同的车道（在这个例子1-4）。标记巷（M）可以染色，成像控制后切割的尺寸。片段大小显示在右侧。 <img src="/files/ftp_upload/2638/2638fig4.jpg" alt="图4" /> 图4。iCLIP使用凝胶电泳PCR扩增cDNA文库的分析。 RNA收回膜（图1）反转录和大小纯化变性凝胶电泳（图2）。三个大小比例的cDNA（高[H +]：[M]：85-120 NT和低[左]：70-85 NT），中型120-200 NT被收回，发送，重新线性和PCR扩增。输入分数的大小不同的结果可以观察到不同的粒度分布的PCR产物。由于PCR引物的cDNA引入76 NT，尺寸范围应161-196新台币为高，中等和146-161粒级低的NT 196-276台币之间。 PCR产物均缺席时没有抗体是免疫（1-3车道）。

<ol>
	<li>Keene, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+regulons:+coordination+of+post-transcriptional+events.">RNA regulons: coordination of post-transcriptional events.</a> Nat Rev Genet. 8, 533-543 (2007).</li><li>Wang, Z., Burge, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splicing+regulation:+from+a+parts+list+of+regulatory+elements+to+an+integrated+splicing+code.">Splicing regulation: from a parts list of regulatory elements to an integrated splicing code.</a> RNA. 14, 802-813 (2008).</li><li>Trifillis, P., Day, N., Kiledjian, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Finding+the+right+RNA:+identification+of+cellular+mRNA+substrates+for+RNA-binding+proteins.">Finding the right RNA: identification of cellular mRNA substrates for RNA-binding proteins.</a> RNA. 5, 1071-1082 (1999).</li><li>Brooks, S. A., Rigby, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+mRNA+ligands+bound+by+the+RNA+binding+protein+hnRNP+A2+utilizing+a+novel+in+vivo+technique.">Characterization of the mRNA ligands bound by the RNA binding protein hnRNP A2 utilizing a novel in vivo technique.</a> Nucleic Acids Res. 28, E49-E49 (2000).</li><li>Tenenbaum, S. A., Carson, C. C., Lager, P. J., Keene, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+mRNA+subsets+in+messenger+ribonucleoprotein+complexes+by+using+cDNA+arrays.">Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays.</a> Proc Natl Acad Sci. 97, 14085-14090 (2000).</li><li>Mili, S., Steitz, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+reassociation+of+RNA-binding+proteins+after+cell+lysis:+implications+for+the+interpretation+of+immunoprecipitation+analyses.">Evidence for reassociation of RNA-binding proteins after cell lysis: implications for the interpretation of immunoprecipitation analyses.</a> RNA. 10, 1692-1694 (2004).</li><li>Ule, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLIP+identifies+Nova-regulated+RNA+networks+in+the+brain.">CLIP identifies Nova-regulated RNA networks in the brain.</a> Science. 302, 1212-1215 (2003).</li><li>Ule, J., Jensen, K., Mele, A., Darnell, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLIP:+A+method+for+identifying+protein-RNA+interaction+sites+in+living+cells.">CLIP: A method for identifying protein-RNA interaction sites in living cells.</a> Methods. 37, 376-386 (2005).</li><li>Licatalosi, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HITS-CLIP+yields+genome-wide+insights+into+brain+alternative+RNA+processing.">HITS-CLIP yields genome-wide insights into brain alternative RNA processing.</a> Nature. 456, 464-469 (2008).</li><li>Yeo, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+RNA+code+for+the+FOX2+splicing+regulator+revealed+by+mapping+RNA-protein+interactions+in+stem+cells.">An RNA code for the FOX2 splicing regulator revealed by mapping RNA-protein interactions in stem cells.</a> Nat Struct Mol Biol. 16, 130-137 (2009).</li><li>Urlaub, H., Hartmuth, K., L&#252;hrmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+two-tracked+approach+to+analyze+RNA-protein+crosslinking+sites+in+native,+nonlabeled+small+nuclear+ribonucleoprotein+particles.">A two-tracked approach to analyze RNA-protein crosslinking sites in native, nonlabeled small nuclear ribonucleoprotein particles.</a> Methods. 26, 170-181 (2002).</li><li>K&#246;nig, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iCLIP+reveals+the+function+of+hnRNP+particles+in+splicing+at+individual+nucleotide+resolution.">iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution.</a> Nat Struct Mol Biol. 17, 909-915 (2010).</li></ol>

，由于iCLIP协议中含有各种各样的酶反应和纯化步骤，它并不总是很容易识别一个实验失败时的问题。为了控制一个或多个阴性对照确定RNA交叉链接网站的特殊性，应保持完整的实验和其后的计算分析。这些控件可无抗体的样品，非交联的细胞，或从基因敲除的细胞或组织免疫。理想的情况下，这些控制实验不应净化任何蛋白- RNA复合物，并因此应该没有信号，SDS - PAGE凝胶和PCR扩增后没有检测到产品。这些控制库的高通量测序应该返回很少有独特的序列。击倒细胞不建议作为时序控制，因为由此产生的序列仍然符合相同的蛋白质，这是小批量击倒细胞纯化的交叉链接网站。 注意事项也应考虑，以避免与以前的实验PCR产物的污染。最大限度地减少这种问题的最好办法是在空间上分开前和后的PCR步骤。理想的情况下，PCR产物和所有后续步骤的分析，应在一个单独的房间内进行。此外，实验室中的每个成员应该使用他们自己的缓冲区和其他试剂。这样可以更容易识别，污染源。

A correction was made to <a href="http://www.jove.com/details.php?id=2638">iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution</a>. There was an error in part 2 of step 3. One of the characters had the incorrect symbol and was corrected to: "...as well as 2 &#x03BC;l Turbo DNase..." instead of: "...as well as 2 ml Turbo DNase..."

A correction to the article iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution was made.

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

凝胶电泳和膜转移，我们建议他XCell SureLock吨使用®迷你细胞和XCell II™印迹模块套件CE标志（Invitrogen公司，EI0002），这是与整个协议中指定使用的不同预制minigels兼容。在协议中提到的所有使用的材料品牌和订单号。在协议中使用的酶的名单列于下表。 <table border="1"><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录＃ </td><td> 评论 </td></tr><tr><td>蛋白质一个磁珠</td><td> Invitrogen公司</td><td> 10001D </td><td>使用鼠标或山羊抗体蛋白G </td></tr><tr><td>核糖核酸我</td><td> Ambion公司</td><td> AM2295 </td><td>活动可以改变批次</td></tr><tr><td> T4 RNA连接酶的我</td><td> NEB </td><td> M0204S </td><td> </td></tr><tr><td> PNK </td><td> NEB </td><td> M0201S </td><td> </td></tr><tr><td>蛋白酶K </td><td>罗氏公司</td><td> 03115828001 </td><td> </td></tr><tr><td>上标III逆转录</td><td> Invitrogen公司</td><td> 18080044 </td><td> </td></tr><tr><td> Circligase二</td><td>震中</td><td> CL9021K </td><td> </td></tr><tr><td> FastDigest ®限制酶BamHI </td><td> Fermentas公司</td><td> FD0054 </td><td> </td></tr><tr><td> AccuPrime™SuperMix我</td><td> Invitrogen公司</td><td> 12342010 </td><td>此PCR混合物为最好的结果，在我们手中</td></tr></table>

iclip - transcriptome-wide mapping of protein-rna interactions with individual nucleotide resolution

RNA结合蛋白的独特的成分和空间布局上的谈话全文（限制性商业惯例）的不同方面的指导后转录调控 1 。因此，对在分子水平上理解转录调控的一个重要步骤是获得关于限制性商业惯例的 2的结合位点的位置信息。 研究蛋白质- RNA的相互作用可以使用生化方法，但这些方法没有解决的RNA在其本土蜂窝范围内具有约束力。研究蛋白- RNA复合物在细胞环境中的初步尝试，采用差异显示或微阵列分析（RIP - CHIP）3-5相结合的亲和纯化或免疫。这些方法很容易识别或间接非生理的相互作用 6 。为了增加特异性和位置的决议，这一战略被称为剪辑（紫外光交联和免疫）7,8。剪辑结合紫外光交联蛋白质和核糖核酸分子，与严格的净化计划，包括变性聚丙烯酰胺凝胶电泳。高通量测序技术相结合，作为一个强大的工具来研究蛋白质- RNA的相互作用，在全基因组规模（简称HITS - CLIP或剪辑，SEQ）9,10剪辑已经证明。近日，PAR - CLIP介绍，使用交联11,12光反应的核苷类似物。 尽管所获得的数据的高特异性，剪辑实验往往产生cDNA文库的有限序列的复杂性。这部分是由于合作纯化的RNA的限制数量和两个低效RNA结扎库准备所需的反应。此外，引物延伸分析表明，许多的cDNA核苷酸交联13提前截断。这种截断cDNA的是失去了在标准的剪辑库准备协议。我们最近开发iCLIP（单个核苷酸分辨率的），它可以捕获截断的cDNA取代低效分子RNA结扎的步骤之一，更有效的分子内的基因函证（图1 ）14。更重要的是，被截断的cDNA测序提供了交叉链接网站的核苷酸决议的立场的见解。我们成功地应用于iCLIP研究核蛋白C的全基因组规模的粒子组织和评估拼接的第 14作用。

Watch this Scientific Journal Video about iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution at JoVE.com

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

成绩单的RNA结合蛋白的空间安排，是一种转录后调控的关键因素。因此，我们制定个人核苷酸分辨率的紫外光交联和免疫（iCLIP），允许精确的全基因组RNA结合蛋白的结合位点的映射。

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 ...

iclip-transcriptome-wide-mapping-protein-rna-interactions-with

Research

JoVE Journal

Biology

58.1K 次观看.  Medical Research Council - MRC. 成绩单的RNA结合蛋白的空间安排，是一种转录后调控的关键因素。因此，我们制定个人核苷酸分辨率的紫外光交联和免疫（iCLIP），允许精确的全基因组RNA结合蛋白的结合位点的映射。

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

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

非侵入性的三维可视化与亚微米分辨率使用同步加速器X射线断层扫描

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

科研

生物学

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and release from individual presynaptic terminals directly, we designed fluorescent false neurotransmitters as substrates for the synaptic vesicle monoamine transporter. Using these probes to image dopamine release in the striatum, we made several observations pertinent to synaptic plasticity. We found that the fraction of synaptic vesicles releasing neurotransmitter per stimulus was dependent on the stimulus frequency. A kinetically distinct "reserve" synaptic vesicle population was not observed under these experimental conditions. A frequency-dependent heterogeneity of presynaptic terminals was revealed that was dependent in part on D2 dopamine receptors, indicating a mechanism for frequency-dependent coding of presynaptic selection.

Hui Zhang and Niko G. Gubernator contributed equally to this work.

A new means to measure neurotransmission optically using fluorescent dopamine analogs.

多巴胺的释放突触前货箱码头在个别的可视化与FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR剪辑 - 一个方法来确定全转录组学，RNA结合蛋白的结合位点

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

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 ...

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

FRET is a process whereby energy is non-radiatively transferred from an excited donor molecule to a ground-state acceptor molecule through long-range dipole-dipole interactions1. In the present sensing assay, we utilize an interesting property of PDA: blue-shift in the UV-Vis electronic absorption spectrum of PDA (Figure 1) after an analyte interacts with receptors attached to PDA2,3,4,7. This shift in the PDA absorption spectrum provides changes in the spectral overlap (J) between PDA (acceptor) and rhodamine (donor) that leads to changes in the FRET efficiency. Thus, the interactions between analyte (ligand) and receptors are detected through FRET between donor fluorophores and PDA. In particular, we show the sensing of a model protein molecule streptavidin. We also demonstrate the covalent-binding of bovine serum albumin (BSA) to the liposome surface with FRET mechanism. These interactions between the bilayer liposomes and protein molecules can be sensed in real-time. The proposed method is a general method for sensing small chemical and large biochemical molecules. Since fluorescence is intrinsically more sensitive than colorimetry, the detection limit of the assay can be in sub-nanomolar range or lower8. Further, PDA can act as a universal acceptor in FRET, which means that multiple sensors can be developed with PDA (acceptor) functionalized with donors and different receptors attached on the surface of PDA liposomes.

We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.

实时监控的配体 - 受体相互作用的荧光共振能量转移

FRET is a process  whereby energy is non-radiatively transferred from an excited donor molecule to  a ground-state acceptor molecule through long-range ...

Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent progress, the functional properties and lateralization of the human auditory cortex are far from being fully understood. Transcranial Magnetic Stimulation (TMS) is a non-invasive technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses, and represents a unique method of exploring plasticity and connectivity. It has only recently begun to be applied to understand auditory cortical function 2. 
An important issue in using TMS is that the physiological consequences of the stimulation are difficult to establish. Although many TMS studies make the implicit assumption that the area targeted by the coil is the area affected, this need not be the case, particularly for complex cognitive functions which depend on interactions across many brain regions 3. One solution to this problem is to combine TMS with functional Magnetic resonance imaging (fMRI). The idea here is that fMRI will provide an index of changes in brain activity associated with TMS. Thus, fMRI would give an independent means of assessing which areas are affected by TMS and how they are modulated 4. In addition, fMRI allows the assessment of functional connectivity, which represents a measure of the temporal coupling between distant regions. It can thus be useful not only to measure the net activity modulation induced by TMS in given locations, but also the degree to which the network properties are affected by TMS, via any observed changes in functional connectivity.
Different approaches exist to combine TMS and functional imaging according to the temporal order of the methods. Functional MRI can be applied before, during, after, or both before and after TMS. Recently, some studies interleaved TMS and fMRI in order to provide online mapping of the functional changes induced by TMS 5-7. However, this online combination has many technical problems, including the static artifacts resulting from the presence of the TMS coil in the scanner room, or the effects of TMS pulses on the process of MR image formation. But more importantly, the loud acoustic noise induced by TMS (increased compared with standard use because of the resonance of the scanner bore) and the increased TMS coil vibrations (caused by the strong mechanical forces due to the static magnetic field of the MR scanner) constitute a crucial problem when studying auditory processing. 
This is one reason why fMRI was carried out before and after TMS in the present study. Similar approaches have been used to target the motor cortex 8,9, premotor cortex 10, primary somatosensory cortex 11,12 and language-related areas 13, but so far no combined TMS-fMRI study has investigated the auditory cortex. The purpose of this article is to provide details concerning the protocol and considerations necessary to successfully combine these two neuroscientific tools to investigate auditory processing. 
Previously we showed that repetitive TMS (rTMS) at high and low frequencies (resp. 10 Hz and 1 Hz) applied over the auditory cortex modulated response time (RT) in a melody discrimination task 2. We also showed that RT modulation was correlated with functional connectivity in the auditory network assessed using fMRI: the higher the functional connectivity between left and right auditory cortices during task performance, the higher the facilitatory effect (i.e. decreased RT) observed with rTMS. However those findings were mainly correlational, as fMRI was performed before rTMS. Here, fMRI was carried out before and immediately after TMS to provide direct measures of the functional organization of the auditory cortex, and more specifically of the plastic reorganization of the auditory neural network occurring after the neural intervention provided by TMS. 
Combined fMRI and TMS applied over the auditory cortex should enable a better understanding of brain mechanisms of auditory processing, providing physiological information about functional effects of TMS. This knowledge could be useful for many cognitive neuroscience applications, as well as for optimizing therapeutic applications of TMS, particularly in auditory-related disorders.

Auditory processing is the basis of speech and music-related processing. Transcranial Magnetic Stimulation (TMS) has been used successfully to study cognitive, sensory and motor systems but has rarely been applied to audition. Here we investigated TMS combined with functional Magnetic Resonance Imaging to understand the functional organization of auditory cortex.

映射后的Theta突发刺激对人类听觉皮层功能成像

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

快速，高效的斑马鱼基因分型采用PCR与高分辨率熔体分析

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

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 ...

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.

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

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 ...

High-resolution Optical Mapping of the Mouse Sino-atrial Node

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The development of novel pharmacological therapies to cure these patients relies on the thorough understanding of both normal physiology and pathophysiology of the SAN. Among the studies of cardiac pacemaking, the mouse SAN is widely used due to its feasibility for modifications in the expression of different genes that encode SAN ion channels or calcium handling proteins. Emerging evidence from electrophysiological and histological studies has also proved the representativeness and similarity of the mouse SAN structure and functions to larger mammals, including the presence of specialized conduction pathways from the SAN to the atrium and a complex pacemakers' hierarchy within the SAN. Recently, the technique of optical mapping has greatly facilitated the exploration and investigation of the origin of excitation and conduction within and from the mouse SAN, which in turn has extended the understanding of the SAN and benefited clinical treatments of SAN dysfunction associated diseases. In this manuscript, we have described in detail how to perform the optical mapping of the mouse SAN from the intact, Langendorff-perfused heart and from the isolated atrial preparation. This protocol is a useful tool to enhance the understanding of mouse SAN physiology and pathophysiology.

Here, we present a protocol for optical mapping of electrical activity from the mouse right atrium and especially the sino-atrial node, at a high spatial and temporal resolution.