Name: enhanced crosslinking immunoprecipitation (eclip) method for efficient identification of protein-bound rna in mouse testis
Uploaded: 2019-05-10T15:00:00
Description: Watch this Scientific Journal Video about Enhanced Crosslinking Immunoprecipitation eCLIP Method for Efficient Identification of Protein-bound RNA in Mouse Testis at JoVE.com

我们感谢埃里克 L 范诺斯特兰德和吉恩·韦奥对原始协议的有益指导。K. z. 得到了中国国家重点研发 & 项目 (2016YFA0500902, 2016YFA0500902) 和中国国家自然科学基金 (31771653) 的支持。L. y. 得到了国家自然科学基金 (81471502, 31871503) 和江苏省创新创业项目的支持。

所有进行的动物实验都得到了南京医科大学委员会的批准。雄性 c57bl6 小鼠在光周期条件下保存, 并提供食物和水。
1. 组织收集和紫外线交联
<ol><li>使用二氧化碳 (CO2) 对2只成年小鼠进行安乐死1-2分钟或直到呼吸停止。接下来, 对每只老鼠进行颈椎脱位。</li>
	<li>在每个免疫沉淀实验中, 从适当年龄的小鼠 (本研究中的一个成体睾丸) 中提取约100毫克的睾丸, 并将组织放?...

<ol>
	<li>Turner, J. M., Mahadevaiah, S. K., Ellis, P. J., Mitchell, M. J., Burgoyne, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pachytene+asynapsis+drives+meiotic+sex+chromosome+inactivation+and+leads+to+substantial+postmeiotic+repression+in+spermatids.">Pachytene asynapsis drives meiotic sex chromosome inactivation and leads to substantial postmeiotic repression in spermatids.</a> Developmental Cell. 10 (4), 521-529 (2006).</li><li>Turner, J. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meiotic+sex+chromosome+inactivation.">Meiotic sex chromosome inactivation.</a> Development. 134 (10), 1823-1831 (2007).</li><li>Kimmins, S., Sassone-Corsi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+remodelling+and+epigenetic+features+of+germ+cells.">Chromatin remodelling and epigenetic features of germ cells.</a> Nature. 434 (7033), 583-589 (2005).</li><li>Ule, J., et al. <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 (5648), 1212-1215 (2003).</li><li>Ule, J., Jensen, K., Mele, A., Darnell, R. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Argonaute+HITS-CLIP+decodes+microRNA-mRNA+interaction+maps.">Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps.</a> Nature. 460 (7254), 479-486 (2009).</li><li>Zheng, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+MOV10L1+associates+with+Piwi+proteins+and+is+an+essential+component+of+the+Piwi-interacting+RNA+(piRNA)+pathway.">Mouse MOV10L1 associates with Piwi proteins and is an essential component of the Piwi-interacting RNA (piRNA) pathway.</a> Proceedings of National Acadamy of Sciences U S A. 107 (26), 11841-11846 (2010).</li><li>Gregersen, L. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MOV10+Is+a+5'+to+3'+RNA+helicase+contributing+to+UPF1+mRNA+target+degradation+by+translocation+along+3'+UTRs.">MOV10 Is a 5' to 3' RNA helicase contributing to UPF1 mRNA target degradation by translocation along 3' UTRs.</a> Molecular Cell. 54 (4), 573-585 (2014).</li><li>Banerjee, S., Neveu, P., Kosik, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+coordinated+local+translational+control+point+at+the+synapse+involving+relief+from+silencing+and+MOV10+degradation.">A coordinated local translational control point at the synapse involving relief from silencing and MOV10 degradation.</a> Neuron. 64 (6), 871-884 (2009).</li><li>Choi, J., Hwang, S. Y., Ahn, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+RNASEH2+and+MOV10+controls+LINE-1+retrotransposition.">Interplay between RNASEH2 and MOV10 controls LINE-1 retrotransposition.</a> Nucleic Acids Research. 46 (4), 1912-1926 (2018).</li><li>Goodier, J. L., Cheung, L. E., Kazazian, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MOV10+RNA+helicase+is+a+potent+inhibitor+of+retrotransposition+in+cells.">MOV10 RNA helicase is a potent inhibitor of retrotransposition in cells.</a> PLoS Genetics. 8 (10), e1002941 (2012).</li><li>Haussecker, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capped+small+RNAs+and+MOV10+in+human+hepatitis+delta+virus+replication.">Capped small RNAs and MOV10 in human hepatitis delta virus replication.</a> Nature Structural &amp; Molecular Biology. 15 (7), 714-721 (2008).</li><li>Kenny, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MOV10+and+FMRP+regulate+AGO2+association+with+microRNA+recognition+elements.">MOV10 and FMRP regulate AGO2 association with microRNA recognition elements.</a> Cell Reports. 9 (5), 1729-1741 (2014).</li><li>Messaoudi-Aubert, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+for+the+MOV10+RNA+helicase+in+Polycomb-mediated+repression+of+the+INK4a+tumor+suppressor.">Role for the MOV10 RNA helicase in Polycomb-mediated repression of the INK4a tumor suppressor.</a> Nature Structural &amp; Molecular Biology. 17 (7), 862-868 (2010).</li><li>Sievers, C., Schlumpf, T., Sawarkar, R., Comoglio, F., Paro, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mixture+models+and+wavelet+transforms+reveal+high+confidence+RNA-protein+interaction+sites+in+MOV10+PAR-CLIP+data.">Mixture models and wavelet transforms reveal high confidence RNA-protein interaction sites in MOV10 PAR-CLIP data.</a> Nucleic Acids Research. 40 (20), e160 (2012).</li><li>Skariah, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mov10+suppresses+retroelements+and+regulates+neuronal+development+and+function+in+the+developing+brain.">Mov10 suppresses retroelements and regulates neuronal development and function in the developing brain.</a> BMC Biology. 15 (1), 54 (2017).</li><li>Zheng, K., Wang, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blockade+of+pachytene+piRNA+biogenesis+reveals+a+novel+requirement+for+maintaining+post-meiotic+germline+genome+integrity.">Blockade of pachytene piRNA biogenesis reveals a novel requirement for maintaining post-meiotic germline genome integrity.</a> PLoS Genetics. 8 (11), e1003038 (2012).</li><li>Vourekas, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNA+helicase+MOV10L1+binds+piRNA+precursors+to+initiate+piRNA+processing.">The RNA helicase MOV10L1 binds piRNA precursors to initiate piRNA processing.</a> Genes &amp; Development. 29 (6), 617-629 (2015).</li><li>Hocq, R., Paternina, J., Alasseur, Q., Genovesio, A., Le Hir, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitored+eCLIP:+high+accuracy+mapping+of+RNA-protein+interactions.">Monitored eCLIP: high accuracy mapping of RNA-protein interactions.</a> Nucleic Acids Research. 46 (21), 11553-11565 (2018).</li><li>Huppertz, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iCLIP:+protein-RNA+interactions+at+nucleotide+resolution.">iCLIP: protein-RNA interactions at nucleotide resolution.</a> Methods. 65 (3), 274-287 (2014).</li><li>Hafner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome-wide+identification+of+RNA-binding+protein+and+microRNA+target+sites+by+PAR-CLIP.">Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP.</a> Cell. 141 (1), 129-141 (2010).</li><li>Hafner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAR-CliP--a+method+to+identify+transcriptome-wide+the+binding+sites+of+RNA+binding+proteins.">PAR-CliP--a method to identify transcriptome-wide the binding sites of RNA binding proteins.</a> Journal of Visualized Experiments. (41), (2010).</li><li>Konig, J., et al. <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> Nature Structural &amp; Molecular Biology. 17 (7), 909-915 (2010).</li><li>Konig, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iCLIP--transcriptome-wide+mapping+of+protein-RNA+interactions+with+individual+nucleotide+resolution.">iCLIP--transcriptome-wide mapping of protein-RNA interactions with individual nucleotide resolution.</a> Journal of Visualized Experiments. (50), (2011).</li><li>Maticzka, D., Ilik, I. A., Aktas, T., Backofen, R., Akhtar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=uvCLAP+is+a+fast+and+non-radioactive+method+to+identify+in+vivo+targets+of+RNA-binding+proteins.">uvCLAP is a fast and non-radioactive method to identify in vivo targets of RNA-binding proteins.</a> Nature Communications. 9 (1), 1142 (2018).</li><li>Van Nostrand, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+transcriptome-wide+discovery+of+RNA-binding+protein+binding+sites+with+enhanced+CLIP+(eCLIP).">Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP).</a> Nature Methods. 13 (6), 508-514 (2016).</li><li>Radulovich, N., Leung, L., Tsao, M. 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随着人们对限制性商业惯例在生物和病理环境中的普遍作用的日益了解, clip 方法被广泛用于揭示限制性商业惯例20、32、33、 34,35。此处描述的协议代表了 eCLIP 方法在鼠标睾丸中的一种适应应用。
在睾丸中执行 eCLIP 的一个挑战是保持新鲜睾丸细胞的活力...

哺乳动物睾丸代表了一个很好的发展模型, 其中一个复杂的细胞分化计划循环运行, 以产生大量的精子。这种模型的一个独特价值在于在精子发生的某些阶段出现转录失活, 通常是在减数分裂性染色体失活 (msci) 发生1,2和圆形精子细胞发生时精子发生时的强烈核压实3。这些连续的转录事件需要转录后基因调控, 其中 rna 结合蛋白 (Rpp) 发挥着至关重要的作用, 塑造转录组和保持男性生育能力。
为了识别个体 rbp 在体内的真正 rna 靶点, 在常规 rna 免疫沉淀 (rip) 6, 7 的基础上, 开发了4,5 交联免疫沉淀 (clip)方法., 通过采用包括紫外线 (UV) 交联、严格清洗和凝胶转移在内的关键步骤, 提高信号特异性。利用 CLIP 与高通量测序相结合的先进应用引起了人们对在全基因组范围内的蛋白质-rna 相互作用进行分析的极大兴趣。除了对 RBP 功能的遗传研究外, 这种确定内源性蛋白质和 RNA 直接相互作用的生化方法对于准确阐明限制性商业惯例的 RNA 调控作用是不可或缺的。例如, MOV10L1 是男性生育能力和 piRNA 相互作用的 RNA (piRNA) 生物发生所需的一种睾丸特异性 RNA 螺旋酶9。它的类似的 mov10 被称为一个无处不在的表达和多功能 rna 螺旋酶, 在 rna 生物学的多个方面的作用10,11,12, 13,14,15,16,17,18. 通过使用传统的 clip-seq, 我们发现 mov10l1 结合和调节初级 pirna 前体, 以启动早期 pirna 处理19,20, 并且 mov10 结合 mrna 3 ' ucr 和非编码 rna在睾丸生殖细胞中的物种 (未显示数据)。
然而, CLIP 最初是一个费力的放射性程序, 然后对库的准备进行排序, 并显著丢失了 CLIP 标签。在传统的 CLIP 中, 使用在两个 RNA 末端结扎的适配器制备 cDNA 文库。蛋白质消化后, 交联的短肽仍附着在 RNA 片段上。这种交联标记部分阻断了 cdna 合成过程中逆转录酶 (rtase) 的进展, 导致约占 cdna 文库21,22的80% 的 cdna 被截断。因此, 只有 RTase 产生的绕过交联位点 (读通) 的 cDNA 片段才会被测序。最近, 各种 CLIP 方法 (如 PAR-CLIP、iCLIP、eCLIP 和 uvCLAP) 被用来识别活细胞中限制性商业惯例的交联位点。PAR-CLIP 涉及365纳米紫外线辐射和光可激活核苷酸类似物的应用, 因此仅适用于培养中的活细胞, 将核苷类似物纳入新合成的转录中容易产生偏差rna 与 23,24蛋白的物理相互作用。在 iCLIP 中, 只有一个适配器连接到交联 RNA 片段的 3个 ' 末端。逆转录酶 (rt) 后, 通过细胞内循环和再线性化获得截断和读通 cDNAs, 然后进行聚合酶链反应 (pcr) 扩增 25,26.然而, 分子内循环的效率相对较低。尽管较旧的 CLIP 协议需要用放射性同位素标记交联 RNA, 但紫外线交联和亲和力纯化 (uvCLAP), 以及严格的串联亲和力纯化过程, 并不依赖于放射性27。然而, uvCLAP 仅限于培养的细胞, 必须通过带有 3x FLAG-HBH 标记的表达载体转染, 以便进行串联亲和力纯化。
在 eCLIP 中, 适配器首先在 RNA 的 3 ' 末端连接, 然后是 rt, 然后在分子间模式下的 3个 cDNAs 极点进行结扎。因此, eCLIP 能够捕获所有截断和读取的 cDNA28。此外, 它既不限于放射性标记, 也不限于根据其原理使用细胞系, 同时保持单核苷酸分辨率。
在这里, 我们提供了适用于鼠标睾丸的 eCLIP 协议的分步说明。简单地说, 该 eCLIP 协议从睾丸小管的紫外线交联开始, 然后使用蛋白质特异性抗体进行部分 RNase 消化和免疫沉淀。接下来, 蛋白质结合 rna 被脱磷酸化, 适配器连接到它的 3 ' 末端。在蛋白质凝胶电泳和凝胶-膜转移后, RNA 通过切割预期大小范围的膜面积进行分离。RT 后, DNA 适配器连接到 cDNA 的 3 ' 端, 然后进行 PCR 扩增。在高通量测序之前对子克隆进行筛选, 作为图书馆的质量控制。该协议能够有效地识别限制性商业惯例的蛋白质结合 RNA 的主要物种, 例如两个表达睾丸的 RNA 螺旋酶 MOV10L1 和 MOV10。

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			<td height="21" style="height:21px;width:229px;">Antibodies</td>
			<td style="width:180px;"></td>
			<td style="width:171px;"></td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:229px;">Anti-mouse MOV10&#160; antibody</td>
			<td style="width:180px;">Proteintech, China</td>
			<td style="width:171px;">10370-1-AP</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:229px;">Anti-mouse MOV10L1 antibody</td>
			<td style="width:180px;">Zheng et al.20109</td>
			<td style="width:171px;">polyclonal antisera UP2175</td>
			<td style="width:224px;">provided by P. Jeremy Wang lab&#65288;University of Pennsylvania&#65289;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">HRP Goat Anti-Rabbit IgG&#160;</td>
			<td style="width:180px;">ABclonal</td>
			<td style="width:171px;">AS014</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Rabbit IgG</td>
			<td style="width:180px;">Beyotime, China</td>
			<td style="width:171px;">A7016</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Equipment</td>
			<td style="width:180px;"></td>
			<td style="width:171px;"></td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">Centrifuge</td>
			<td style="width:180px;">Eppendorf, Hamburg, Germany</td>
			<td style="width:171px;">5242R</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Digital sonifier</td>
			<td style="width:180px;">BRANSON,USA</td>
			<td style="width:171px;">BBV12081048A</td>
			<td style="width:224px;">450 Watts; 50/60 HZ</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">DynaMag-2 Magnet</td>
			<td style="width:180px;">Invitrogen,USA</td>
			<td style="width:171px;">12321D</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Mini Blot Module</td>
			<td style="width:180px;">Invitrogen,USA</td>
			<td style="width:171px;">B1000</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Mini Gel Tank</td>
			<td style="width:180px;">Invitrogen,USA</td>
			<td style="width:171px;">A25977</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shaking incubator</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>Thermomixer comfort&#160;</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Tissue Grinder, Dounce</td>
			<td>PYREX, USA</td>
			<td>1234F35</td>
			<td style="width:224px;">only&#160; the &#34;loose&#34; pestle is used in this protocol</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">TProfessional standard 96 Gradient</td>
			<td style="width:180px;">Biometra, Germany</td>
			<td style="width:171px;">serial no.: 2604323</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Tube Revolver&#160;</td>
			<td style="width:180px;">Crystal, USA</td>
			<td style="width:171px;">serial no.: 3406051</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">UV-light cross-linker</td>
			<td>UVP, USA</td>
			<td style="width:171px;">CL-1000</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Materials</td>
			<td></td>
			<td style="width:171px;"></td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">TC-treated Culture Dish</td>
			<td>Corning, USA</td>
			<td style="width:171px;">430167</td>
			<td style="width:224px;">100 mm&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Tubes</td>
			<td style="width:180px;">Corning, USA</td>
			<td style="width:171px;">430791</td>
			<td style="width:224px;">15 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Microtubes tubes</td>
			<td style="width:180px;">AXYGEN , USA</td>
			<td style="width:171px;">MCT-150-C</td>
			<td style="width:224px;">1.5 mL&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Reagents&#160;</td>
			<td style="width:180px;"></td>
			<td style="width:171px;"></td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:229px;">Acid phenol/chloroform/isoamyl alcohol&#160;</td>
			<td style="width:180px;">Solarbio, China</td>
			<td style="width:171px;">P1011</td>
			<td style="width:224px;">25:24:01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">AffinityScript Enzyme</td>
			<td style="width:180px;">Agilent, USA</td>
			<td style="width:171px;">600107</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Antioxidant</td>
			<td style="width:180px;">Invitrogen,USA</td>
			<td style="width:171px;">NP0005</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:229px;">DH5&#945; competent bacteria</td>
			<td style="width:180px;">Thermo Scientific, USA</td>
			<td>18265017</td>
			<td style="width:224px;">these economical cells yield &#62;1 x 106 transformants/&#181;g control DNA per 50 &#181;L reaction.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">DMSO</td>
			<td style="width:180px;">Sigma-Aldrich, USA</td>
			<td style="width:171px;">D8418</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">DNA Ladder</td>
			<td style="width:180px;">Invitrogen, USA</td>
			<td style="width:171px;">10416014</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">dNTP</td>
			<td style="width:180px;">Sigma-Aldrich, USA</td>
			<td style="width:171px;">DNTP100-1KT</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dynabeads Protein A&#160;</td>
			<td>Invitrogen, USA</td>
			<td>10002D</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ECL reagent</td>
			<td>Vazyme, China</td>
			<td>E411-04</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">EDTA</td>
			<td>Invitrogen, USA</td>
			<td style="width:171px;">AM9260G</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">EDTA free protease inhibitor cocktail</td>
			<td style="width:180px;">Roche, USA</td>
			<td style="width:171px;">04693132001</td>
			<td style="width:224px;">add fresh</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Exo-SAP-IT</td>
			<td style="width:180px;">Affymetrix, USA</td>
			<td style="width:171px;">78201</td>
			<td style="width:224px;">PCR Product Cleanup Reagent&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">FastAP enzyme</td>
			<td style="width:180px;">Thermo Scientific, USA</td>
			<td style="width:171px;">EF0652</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">LDS Sample Buffer</td>
			<td style="width:180px;">Thermo Scientific, USA</td>
			<td style="width:171px;">NP0007</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">MetaPhor Agarose&#160;</td>
			<td style="width:180px;">lonza, Switzerland</td>
			<td style="width:171px;">50180</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:229px;">MgCl2</td>
			<td>Invitrogen, USA</td>
			<td style="width:171px;">AM9530G</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:229px;">MiniElute gel Extraction</td>
			<td style="width:180px;">QIAGEN, Germany</td>
			<td style="width:171px;">28604</td>
			<td style="width:224px;">column store at 4 &#8451;; buffer QG=gel dissolving buffer; buffer PE= wash buffer(for step 14)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">MyONE Silane beads</td>
			<td style="width:180px;">Thermo Scientific, USA</td>
			<td>37002D</td>
			<td style="width:224px;">nucleic acids extraction magnetic beads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Invitrogen,USA</td>
			<td>AM9759 
			&#160;</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">NP-40</td>
			<td style="width:180px;">Amresco, USA</td>
			<td style="width:171px;">M158-500ML</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">NuPAGE Bis-Tris Protein Gels</td>
			<td style="width:180px;">Invitrogen, USA</td>
			<td style="width:171px;">NP0336BOX</td>
			<td style="width:224px;">4%&#8211;12%,1.5 mm, 15-well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">NuPAGE MOPS SDS Buffer Kit&#160;</td>
			<td style="width:180px;">Invitrogen, USA</td>
			<td style="width:171px;">NP0050</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Gibco, USA</td>
			<td>10010023</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">Phase-Locked Gel (PLG) heavy tube&#160;&#160;</td>
			<td style="width:180px;">TIANGEN, China</td>
			<td style="width:171px;">WM5-2302831</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">PowerUp SYBR Green Master Mix</td>
			<td style="width:180px;">Applied Biosystems, USA</td>
			<td style="width:171px;">A25742</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">proteinase K</td>
			<td style="width:180px;">NEB, New England&#160;</td>
			<td style="width:171px;">P8107S</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Q5 PCR master mix&#160;</td>
			<td style="width:180px;">NEB, New England&#160;</td>
			<td style="width:171px;">M0492L</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">RLT buffer</td>
			<td style="width:180px;">QIAGEN, Germany</td>
			<td style="width:171px;">79216</td>
			<td style="width:224px;">RNA purification lysis buffer&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">RNA Clean &#38; Concentrator-5 columns&#160;</td>
			<td style="width:180px;">ZYMO RESEARCH, USA</td>
			<td style="width:171px;">R1016</td>
			<td style="width:224px;">RNA purification and concentration columns</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">RNase I&#160;</td>
			<td>Invitrogen, USA</td>
			<td style="width:171px;">AM2295</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">RNase Inhibitor</td>
			<td style="width:180px;">Promega, USA</td>
			<td style="width:171px;">N251B</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">RQ1 DNase</td>
			<td style="width:180px;">Promega,USA</td>
			<td style="width:171px;">M610A</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Sample Reducing Agent</td>
			<td>Invitrogen,USA</td>
			<td style="width:171px;">NP0009</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">SDS Solution</td>
			<td>Invitrogen, USA</td>
			<td style="width:171px;">15553027</td>
			<td style="width:224px;">10%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Sodium deoxycholate</td>
			<td style="width:180px;">Sigma-Aldrich, USA</td>
			<td style="width:171px;">30970</td>
			<td style="width:224px;">protect from light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">T4 PNK enzyme</td>
			<td style="width:180px;">NEB, New England&#160;</td>
			<td style="width:171px;">M0201L</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">T4 RNA ligase 1 high conc</td>
			<td style="width:180px;">NEB, New England&#160;</td>
			<td style="width:171px;">M0437M</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">TA/Blunt-Zero Cloning Mix</td>
			<td style="width:180px;">Vazyme, China</td>
			<td style="width:171px;">C601-01</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">TBE</td>
			<td>Invitrogen,USA</td>
			<td style="width:171px;">AM9863</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCI Buffer</td>
			<td>Invitrogen, USA</td>
			<td>15567027</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Triton X-100</td>
			<td style="width:180px;">Sangon Biotech, China</td>
			<td style="width:171px;">A600198&#160;</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Tween-20</td>
			<td style="width:180px;">Sangon Biotech, China</td>
			<td style="width:171px;">A600560</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Urea</td>
			<td style="width:180px;">Sigma-Aldrich, USA</td>
			<td style="width:171px;">U5378</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">X-ray Films</td>
			<td style="width:180px;">Caresteam, Canada</td>
			<td style="width:171px;">6535876</td>
			<td style="width:224px;"></td>
		</tr>
	</tbody>
</table>

enhanced crosslinking immunoprecipitation (eclip) method for efficient identification of protein-bound rna in mouse testis

精子发生定义了哺乳动物雄性生殖细胞分化的高度有序过程。在睾丸中, 转录和翻译是不耦合的, 这突出了限制性商业惯例组织的转录后基因表达调控的重要性。为了阐明 RBP 的机械作用, 可以使用交联免疫沉淀 (CLIP) 方法来捕获其内源性直接 RNA 靶点并定义实际的相互作用位点。增强的 CLIP (eCLIP) 是一种新开发的方法, 与传统的 Clip 相比具有多种优势。然而, 到目前为止, 电子浓缩公司的使用仅限于细胞系, 这就需要扩展应用程序。在这里, 我们使用 eCLIP 在鼠标睾丸中研究 MOV10 和 MOV10L1 这两个已知的 rna 结合螺旋。正如预期的那样, 我们发现 MOV10 主要与3个未翻译的 mRNA 区域 (Ucr) 结合, MOV10L1 有选择地与 piRNA 相互作用的 RNA (piRNA) 前体转录结合。我们的 eCLIP 方法允许通过对亚克隆进行小规模测序, 从而提供合格的库, 快速确定受各种限制性商业惯例约束的主要 RNA 物种, 作为进行深度测序的理由。本研究为哺乳动物睾丸的电子规则规则制定了适用的依据。

Eclip 过程和结果如图 1、 图 2、 图 3、 图 4所示。用二氧化碳对小鼠进行安乐死, 用手术剪刀在下腹部做了一个小切口 (图 2a,b)。鼠标睾丸研磨后被切除、失谐, 然后 uv 交?...

Watch this Scientific Journal Video about Enhanced Crosslinking Immunoprecipitation eCLIP Method for Efficient Identification of Protein-bound RNA in Mouse Testis at JoVE.com

Enhanced Crosslinking Immunoprecipitation eCLIP Method for Efficient Identification of Protein-bound RNA in Mouse Testis

增强交联免疫沉淀 (eCLIP) 方法, 有效鉴定小鼠睾丸蛋白质结合 rna

在这里, 我们提出了一个 eCLIP 协议, 以确定主要的 RBP 候选在睾丸的 rna 目标。

Spermatogenesis defines a highly ordered process of male germ cell differentiation in mammals. In testis, transcription and translation are uncoupled, ...

与RNA结合的蛋白质对细胞生理学至关重要。重要的是，RBP在整个精子生成过程中都表达得非常强烈，并且在生殖细胞的所有阶段都作为重要的转录后调节器被很好地记录。确定绑定目标对于阐明 RBP 的机械作用至关重要。该协议代表了黄道针方法的适应应用，用于捕获内源性直接RNA靶点，并建立了在哺乳动物睾丸中黄热化的适用基础。此方法的主要优点是，它是非放射性的，时间密集程度更低，并提供更强的信号噪声比，因为大小匹配输入将作为真实目标的适当背景。最后，我们认为亚组酮的小规模测序对于深度测序是有用的。演示程序将是徐秋实，我的同事。为了开始这个程序，从每个免疫沉淀实验中从适当年龄的小鼠身上收获大约100毫克的睾丸。将组织放在冰冷的 PBS 中。使用一对细尖的钳子，轻轻取出图尼卡阿尔布吉纳。在组织研磨机中加入三毫升冰冷的PBS，并使用松玻璃用温和的机械力将组织三联。接下来，将组织悬浮液转移到细胞培养皿中，并加入多达六毫升的冰冷PBS。快速摇动盘子，使液体均匀地覆盖盘子底部。在254纳米时，将悬浮在冰上三次，每厘米400毫焦耳。确保将悬浮液混合在每次辐照之间。然后，在15毫升锥形管中收集悬浮液，并在1，200倍g和4度下离心5分钟。取出上一液，将颗粒重新在一毫升PBS中。在此之后，将悬浮液转移到1.5毫升离心管。在1000倍的g和4摄氏度的离心2分钟，并丢弃上一代。首先，将每个样品的125微升蛋白质 A 磁珠添加到新鲜的离心管中。将管子放在磁铁上，将珠子与溶液分离。10秒后，取出上清液，用一毫升冰冷解液缓冲液洗涤珠子两次。将珠子用10微克的黄心抗体重新填充在100微升的冷解解缓冲液中。在室温下旋转管45分钟。然后，用一毫升的冰冷解液缓冲液清洗珠子两次。首先，将组织颗粒重新泵中一毫升的冷解液缓冲液中，其中含有22微升50倍无EDTA蛋白抑制剂鸡尾酒和11微升RNase抑制剂。在冰上保持 15 分钟的样品。对文本协议中概述的每个样本进行声波化。接下来，在每个管中加入四个微升的DNase，并混合好。在 37 摄氏度下孵育 10 分钟，同时在转速 1200 rpm 时摇晃。在此之后，加入10微升稀释的RNase，混合好。在37摄氏度下孵育5分钟，同时在1200转/小时摇晃。然后，在15，000倍g和4摄氏度的离心机20分钟，以清除酸盐。仔细收集上一液，并保存 RWB 和 RRI 样品的输入样本。首先，在准备好的珠子中加入一毫升的落酸，在四摄氏度下旋转样品两小时或一夜。在此之后，用磁性支架收集珠子，并丢弃上经剂。用900微升高盐缓冲液清洗珠子两次，然后用900微升的洗涤缓冲液洗涤珠子两次。然后，用500微升1倍脱磷缓冲液清洗珠子一次。首先，丢弃上清液，并使用细移液器尖端去除任何残留液体。在每个样品中加入25个三主要结扎主混合物的微升，并小心地将移液器混合。在每个样品中加入2.5微升RNA适配器X1A和2.5微升RNA适配器X1B。通过移液或轻拂小心混合。在25摄氏度下孵育75分钟，确保轻拂管混合每10分钟。用500微升冷洗缓冲液清洗珠子一次。接下来，用500微升的冷高盐缓冲液洗一次珠子，然后用500微升的冷洗缓冲液清洗珠子。重复这两个洗涤再次。磁性分离珠子，并使用精细移液器尖端去除任何残留液体。将珠子重新填充在 100 微升冷洗缓冲液中，然后将 20 微升作为 RWB 样品移动到新管中。将 7.5 微升 4x LDS 样品缓冲液和 3 个 10x 样品还原剂微升添加到其余样品中。在 70 摄氏度下孵育 10 分钟，同时在 1200 rpm 时摇晃。之后，在冰上冷却样品一分钟，在1000次g和4摄氏度下离心1分钟。对于 RWB 凝胶，将管子放在磁铁上，将蛋白质从珠子中分离出。将 15 微升样品加载到每个井中。接下来，在500毫升的1xSDS运行缓冲液中加入500微升的抗氧化剂。以 200 伏、1 倍 SDS 运行缓冲液 50 分钟或直到染料前部底部运行凝胶。将蛋白质-RNA复合物从凝胶转移到硝基纤维素膜中，在10伏特的1x转移缓冲液中，用10%甲醇将70分钟。在室温下将 5%牛奶中的 RWB 膜在 TBST 中阻塞一小时。在TBST中冲洗膜，并在4摄氏度下用TBST中的原抗体孵育。在此之后，用 TBST 洗两次，每次洗涤持续五分钟。在室温下用二次抗体在TBST中孵育一小时。然后，用TBST清洗膜三次。接下来，混合等量的ECL缓冲液 A 和缓冲液 B.将这种混合物添加到膜中，孵育一分钟。用塑料包装盖住膜，在室温下将其暴露在 X 射线膜上两到三分钟，然后再开发薄膜。在这项研究中，MOV10黄道从成年野生型小鼠与RNase I治疗交联的利萨酸盐，目标蛋白，这是约114千吨，成功地丰富。从各种样品中稀释一至十的cDNA的qPCR结果表明，非交联样品的RNA回收率下降。观察到非交联组的 Ct 值通常比 UV 交联组的五倍以上。此处显示了通过气糖凝胶电泳进行PCR扩增和尺寸选择具有代表性的结果。底向-二丁车产品出现在大约 140 个基对上。两个代表性子束序列的 UCSC 基因组浏览器视图显示，MOV10 绑定的黄道卡标签位于基因 Fto 的三主 UTR 内。三等 UTR 目标的近似速率为 75%，与 HEK293 细胞和睾丸中的大多数 MOV10 目标一致。相比之下，MOV10L1绑定的黄道标记位于piRNA聚类内，指示MOV10L1目标piRNA前体。piRNA前体靶目标的近似率为42%，这反映了以往研究的趋势。MOV10L1 转点与 40 单位每毫升 RNase I 消化产生相对更多的序列，小于 20 个碱基对。此处描述的此协议代表了黄道方法在复制中的采用，而这一领域RNA-RBP相互作用知识相当不足。

Research

JoVE Journal

Biology

Enhanced Crosslinking Immunoprecipitation (eCLIP) Method for Efficient Identification of Protein-bound RNA in Mouse Testis

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

使用早期的小鼠胚胎组织特异性基因的染色质免疫沉淀检测

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

科研

生物学

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

高效的小鼠胚胎干细胞分化成运动神经元

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification

进行阴道灌洗，结晶紫染色法和阴道细胞学评价小鼠发情周期分期鉴定

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the ...

Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

生物质的使用限制金额高效的染色质免疫沉淀

Chromatin immunoprecipitation (ChIP) is a  widely-used method for determining the interactions of different proteins with  DNA in chromatin of living ...

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

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 ...

In Vivo Microinjection and Electroporation of Mouse Testis

In Vivo Microinjection and Electroporation of Mouse Testis

在小鼠睾丸活体显微注射和电穿孔

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular ...

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

增强Northern印迹检测小RNA种类的<em&gt;果蝇</em

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular ...

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

小RNA分子的高效结扎的微小RNA定量高通量测序

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

染色质免疫沉淀拟南芥蛋白-DNA相互作用的鉴定<em&gt;在体内</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

tchip-seq: 细胞专用表观体分析

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...