我们感谢T.红，三菱商事。蔡O.庄园，E.西格尔，M.黑田东彦，T. Swigut，一讨论Shestopalov。新加坡（CC），国立卫生研究院以R01 CA118750和以R01-HG004361（HYC），加州再生医学研究所（HYC）科学，技术和研究机构的支持。 HYC是霍华德·休斯医学研究所的早期职业科学家。

1。探针设计 CHIRP设计反义DNA瓦片目标RNA的选择性检索的探针。 <ol><li>设计反义寡核苷酸探针在网上探头设计师<a href="http://singlemoleculefish.com">singlemoleculefish.com</a> 18。 </li><li>使用这些参数：探头= 1探针/ 100 bp的RNA的长度; 2）目标GC％= 45; 3）寡核苷酸长度= 20; 4）间隔长度= 60-80。如果太长的设计师闯入段的RNA。省略重复或广泛的同源性区域。 </li><li>为了反义DNA探针在3总理结束BiotinTEG。 </li><li>根据自己的位置沿RNA标记探针。他们分成两个游泳池，使“甚至”池包含所有探头的编号2，4，6，等“奇”池包含探针编号1，3，5，等到100微米的浓度和稀释的探针池储存于-20°C </li><li>所有的实验都进行两个游泳池，对方的内部控制。真正的RNA依赖的信号会从目前两个池，而探头的噪音会是唯一的每个池。这适用于CHIRP-qPCR和啁啾SEQ。 </li></ol> 2。收获细胞 收集细胞CHIRP实验将使用。 <ol><li>生长在细胞组织培养板或汇合瓶。冲洗用磷酸盐缓冲液（PBS）和t​​rypsinize。淬火胰蛋白酶与媒体的2倍量，吸起来，撞出细胞和悬浮成单细胞悬液。所有媒体和再悬浮细胞转移到50毫升猎鹰管。 20亿个细胞通常足够一CHIRP样品。 </li><li>在800RCF 4分钟旋转细胞。抽吸媒体和40毫升的PBS重悬40万个细胞，如果有必要结合管。在800RCF 4分钟旋转细胞。倒出PBS，小心吸的角度上剩余的液体。 </li></OL&gt; 3。交联细胞，收集细胞沉淀 交联戊二醛保存RNA的染色质相互作用，并准备细胞沉淀收集细胞。 <ol><li>在室温下执行的所有步骤。 </li><li> 1％的戊二醛在室温PBS准备。准备10毫升，每10万细胞（0.4毫升25％戊二醛股票+ 9.6毫升PBS）。戊二醛必须使用新鲜。 </li><li>点选底部猎鹰管撞出颗粒。重悬细胞沉淀在1％的戊二醛，具有体积小，以避免块开始，然后补足全量。反相混合。在室温下10分钟架桥月底到年底摇床或旋转。 </li><li>快速为5分钟，在室温下甘氨酸1.25米的十分之一体积的交联反应。 </li><li>在2000RCF离心5分钟。冷藏20毫升PBS一次，在2000RCF纺纱5分钟，吸上清和洗颗粒。 </li><li>吸和悬浮在W灰化，交联颗粒与1毫升冷PBS每20万个细胞。每毫升转移到Eppendorf管和自旋为3分钟，4℃枪头删除尽可能多的PBS尽可能仔细2000RCF。 </li><li>闪光冻结在-80在液氮和存储单元颗粒°C间下去。 </li></ol> 4。细胞裂解 裂解交联细胞准备细胞裂解。 <ol><li>在室温下解冻冷冻细胞颗粒。点击驱逐和混合细胞沉淀。自旋向下沉淀在2000RCF 3分，在4°C用锋利的10μL枪头，以消除任何剩余的PBS。 </li><li>在电子天平（精确到1毫克）皮的空Eppendorf管的质量（我们管重1.060克，非常一致）。权衡每个颗粒，并记录其重量。交联的HeLa细胞整整15厘米的菜通常重100毫克。 </li><li>补编裂解液（10X球团矿质量，如1毫升100毫克）与FRESĤ蛋白酶抑制剂PMSF和Superase中（见附件缓冲区列表）。拌匀。 </li><li> 10X量补充裂解液加入每管和悬浮颗粒。对于小颗粒&lt;25毫克，250μL补充裂解液中悬浮。暂停应光滑。如果没有，分为500μL等分的悬挂和使用机动颗粒搅拌机打破团块。立即着手超声。 </li></ol> 5。超声 通过超声分散交联细胞裂解物的剪切DNA的。 <ol><li>超声在15毫升猎鹰管细胞裂解Bioruptor。使用&lt;1.5毫升裂解，每管和更快的超声，超声时间不超过两管。 </li><li>超声在4℃最高设置30秒的水洗澡ON，OFF脉冲间隔4​​5秒。检查裂解液每30分钟。继续超声分散，不再浑浊，直到细胞裂解。这可能要花费少则30分钟，多达4个小时。数量管，样本量，水浴温度，超声时间内将影响这个过程需要多久。超声管可能会在不同的利率，使他们凝聚在一起，每30分钟重新分配到原管，确保同质化。注：戊二醛交联细胞采取显着更长的时间比甲醛等值超声。 </li><li>当裂解变为清晰，5μL的裂解转移到一个新的Eppendorf管中。加入90μL的DNA蛋白酶K（PK），缓冲区（缓冲区列表）和5μL的PK。旋涡混合后短暂离心。孵育45分钟，在50°C。 </li><li> Qiagen公司的PCR纯化试剂盒提取的DNA。洗脱在30μL的Qiagen公司的洗脱缓冲液（EB）的DNA和1％琼脂糖凝胶电泳检查DNA的大小。如果大量的DNA涂抹100-500基点，超声是完整的。如果没有，继续超声。 </li><li>离心10分钟的超声波处理样品在16100RCF 4°C。结合上清，分装成1毫升样品和闪光冻结液体nitrogeN。储存在-80°C。 </li></ol> 6。 CHIRP 杂交生物素标记的DNA探针，RNA和隔离结合的染色质。 <ol><li>在室温解冻的染色质管。 </li><li>准备杂交缓冲区（缓冲区列表，准备染色毫升，每2毫升）。涡旋混合。 </li><li>对于一个典型的的CHIRP使用裂解液1毫升的样品，取出10μLRNA输入和10μL的Eppendorf管中的DNA INPUT和地方。保持在冰上，直到进一步利用。 </li><li> 1毫升染色质转移至15毫升的猎鹰管。杂交缓冲液中加入2毫升每管。总体积为&lt;1.5毫升，使用Eppendorf管。 </li><li>在室温下解冻探头。检查数量，如果你不使用它在很长一段时间（100微米探针的NanoDrop探头规格〜500-600 ng /μl的单链DNA的设置）。具体管（100 pmol探针染色每1毫升，1μL100 pmol /μL的每1毫升染色探头）探头适当的音量。拌匀。在37℃孵育4小时用颤抖。 </li><li>随着杂交余下的20分钟，准备的C-1磁珠（储存在4°C）。使用100μL的探头，每100 pmol。 1毫升unsupplemented裂解缓冲液洗三次，使用从缓冲区的DynaMag-2磁铁带单独的珠子。 </li><li>原体积的裂解液重悬珠;与新鲜PMSF，有价证券和Superase在补充。经过4小时的杂交反应完成后，每管加100μL珠。拌匀。在37°C孵育30分钟用颤抖。 </li><li>准备洗净的缓冲区，每个样品（5毫升）。涡旋混合。预温至37°C。使用前加入PMSF。 </li><li>用1毫升洗涤缓冲五倍珠。在第一次洗，使用-15 DynaMag磁条单独的珠子，倒出，并重新悬浮在洗涤缓冲液1毫升。传输量1.5 mL的Eppendorf管中。在37°C孵育5分钟摇晃。 </li><li>在随后的洗涤，离心管上每一个minicentrifuge，设置的DynaMag-2 1分钟的磁条上的样本。倒出样品，在洗涤缓冲液1毫升悬浮1 Kimwipe的，任何水滴擦拭。在37°C孵育5分钟摇晃。重复5个总清洗。 </li><li>在最后一次洗涤，悬浮珠。取出100μL和RNA分离作废。储备900μLDNA的分数。放在DynaMag-2磁条所有的管子和删除洗缓冲区。后短暂离心管放在磁铁带。用锋利的10μL枪头完全删除最后的洗涤缓冲位。 </li></ol> 7。 RNA分离 QRT-PCR定量从CHIRP样本中提取的RNA部分。 <ol><li>以100μL，珠样品和10μLRNA输入采样。加入85μL的RNA PK缓冲液pH 7.0的RNA输入。在95μL，RNA的PK缓冲液pH 7.0重悬珠。在50°C加入5μL，Proteine​​ase K和孵育45分钟月底到年底晃动。 </li><li>简单地降速所有的管子，煮沸10分钟的热块样品在95°C。 </li><li>冷藏在冰样品，加入500μL的TRIzol试剂，涡旋大力，持续10秒。在室温下孵育10分钟。贮存于-80°C或继续执行步骤4。 </li><li>加入100μL氯仿TRIZOL处理的样品。涡大力，持续10秒。在4°C为15分钟的台式离心机上旋转在16100RCF </li><li>删除〜400μL水上清液，避免有机和接口。 </li><li>加入600μL（1.5卷）100％的乙醇，并拌匀。旋转样品通过MIRNeasy迷你列。与RWT的（MIRNeasy迷你包），每制造商的协议的视网膜色素上皮2X 1X洗。与30μL无核酸酶H 2 O（NFH 2）洗脱。 </li><li>对待每制造商的协议无 DNA的RNA洗脱液。反应完成后，样本加热15分钟，在65°C至完全灭活任何剩余的DNA酶。 </li><li>使用1μL，RNA的分离定量RT-PCR分析，每口井，以确认lncRNA检索。 GAPDH的经常被用来作为阴性对照。 </li></ol> 8。 DNA提取 从啁啾样品中提取DNA的部分，经测序，以确定或由定量PCR定量。 <ol><li>准备DNA洗脱缓冲液（见缓冲区列表），每个样品150μL，包括DNA输入。 </li><li>新增10μLRNA酶A（10毫克/毫升）和每毫升DNA洗脱缓冲液10μL，核糖核酸酶H（10 U /μL），涡旋混合。 </li><li>悬浮每个样品150μL核糖核酸酶与DNA洗脱缓冲液珠。 （重悬在140μLDNA输入）在37℃孵育30分钟用颤抖。 </li><li>单独的珠子和上清DynaMag-2磁条。除去上清液，加入标记的试管。 </li><li>准备第二等份的DNA洗脱缓冲液10μL的RNA酶A（10毫克/毫升）和，RNaseH（10的U /μL）8.2完全一样做）。加入150μL，每个样品（包括DNA输入），孵育，弃上清。收集所有的上清液（建议立即进行删除D是〜300μL）。 </li><li>加入15μLPK每个样品。在50°C孵育45分钟用颤抖。 </li><li>前降速黄色锁相凝胶管（5PRIME）的。锁相凝胶管转移的DNA样本，并添加300μLPhOH：每个样品异戊：氯仿。大力摇晃10分钟，并在16100RCF 5分钟的台式离心机上旋转，在4°C采取水从顶部（约300μL）。新增3 GlycoBlue微升，30μL的醋酸钠，和900μL100％的乙醇。混合和储存在-20°C过夜。 </li><li>旋转16100RCF样品在4°C. 30分钟</li><li>小心倒出上清液。加入1毫升70％乙醇，涡旋混合。在16100RCF离心5分钟。取出上清液，用吸管。空气干燥1分钟。悬浮在30μL的光大。 </li><li> DNA样本分析每Illumina的协议的高通量测序库的qPCR或准备就绪。 </li></ol> 10。代表结果 图1 </stroNG&gt;描述啁啾的工作流程。通常一个成功的CHIRP实验丰富大大超过非特定的RNA靶RNA。 图2显示了人类端粒酶RNA（TERC）从HeLa细胞中GAPDH的，丰富的，可作为阴性对照细胞RNA的富集。执行CHIRP拉低TERC的RNA（〜88％）在细胞中的大部分，而只有0.46％的GAPDH RNA检索，展示〜200倍的富集因子。针对LacZ基因的RNA，这是不是在哺乳动物细胞中表达（ 图2），探针，如非特异性探针，可以用来作为额外的阴性对照。 通常富集负地区的qPCR测量时，预计到绑定目标lncRNA的DNA区域。 图3显示四个热风的方向在小学人包皮成纤维细胞的网站，我们通过在同一细胞系的CHIRP SEQ中确定的定量PCR验证，而TERC和GAPDH的DNA位点本身RVE作为阴性对照地区。两个“甚至”和“奇”探头设置产生了负面的地区，真正的lncRNA结合位点的一个标志，预计热风绑定网站可比富集。 啁啾丰富的DNA高通量测序产生的全球地图lncRNA结合位点。 果蝇的lncRNA roX2是与X染色体交互方式在剂量补偿的要求。 图4显示了一个X染色体的部分roX2约束力的文件。两个“甚至”和“奇”的样品已测序和其独特的声音已被淘汰，产生重叠信号的轨道。每一个“高峰期”，在这里表示强烈的roX2约束力的网站。完整的跟踪和列表roX2靶基因已在楚等人2011 17。 <img alt="图1。" src="/files/ftp_upload/3912/3912fig1.jpg" /> 图1。的CHIRP多项式的流程图dure。染色质交联lncRNA： 在体内生物素蛋白加合物平铺探测是杂交lncRNA目标，和染色质复合物使用磁链亲和素磁珠纯化，通过严格的清洗。我们洗脱lncRNA结合的DNA或蛋白质的核糖核酸酶A和H鸡尾酒lncRNA一个假定的结合序列，在橙系统化。 2011年以前在楚等出版。17 <img alt="图2。" src="/files/ftp_upload/3912/3912fig2.jpg" /> 图2。CHIRP丰富，为人类TERC的RNA。 TERC asDNA探头撷取〜蜂窝TERC RNA不到GAPDH的88％。 LacZ基因的asDNA探针作为阴性对照和检索既不的RNA。平均值±SD所示。 2011年以前在楚等出版。17 <img alt="图3。" src="/files/ftp_upload/3912/3912fig3.jpg" /> 图3。热风CHIRP-qPCR的首要人权埃斯金的成纤维细胞。NFKBIA，HOXD3-4，SERINC5和ABCA2的与热风进行交互的地区。TERC和GAPDH服务作为阴性对照。平均值±SD所示。 2011年以前在楚等出版。17 <img alt="图4。" src="/files/ftp_upload/3912/3912fig4.jpg" /> 图4。CHIRP roX2的RNA-seq的数据在SL2 果蝇细胞。 “即使”和“奇”分别进行测序，他们的数据合并，以反映都只有普通的山峰。合并后的轨道。 2011年以前在楚等出版。17

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+noncoding+RNA+as+modular+scaffold+of+histone+modification+complexes.">Long noncoding RNA as modular scaffold of histone modification complexes.</a> Science. 329, 689-693 (2010).</li><li>Zappulla, D. C., Cech, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+as+a+flexible+scaffold+for+proteins:+yeast+telomerase+and+beyond.">RNA as a flexible scaffold for proteins: yeast telomerase and beyond.</a> Cold Spring Harb. Symp. Quant. Biol.. 71, 217-224 (2006).</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=The+Air+noncoding+RNA+epigenetically+silences+transcription+by+targeting+G9a+to+chromatin.">The Air noncoding RNA epigenetically silences transcription by targeting G9a to chromatin.</a> Science. 322, 1717-1720 (2008).</li><li>Carter, D., Chakalova, L., Osborne, C. S., Dai, Y. F., Fraser, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-range+chromatin+regulatory+interactions+in+vivo.">Long-range chromatin regulatory interactions in vivo.</a> Nature. 32, 623-626 (2002).</li><li>Chu, C., Qu, K., Zhong, F. L., Artandi, S. E., Chang, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+Maps+of+Long+Noncoding+RNA+Occupancy+Reveal+Principles+of+RNA-Chromatin+Interactions.">Genomic Maps of Long Noncoding RNA Occupancy Reveal Principles of RNA-Chromatin Interactions.</a> Mol. Cell. , (2011).</li><li>Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A., Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+individual+mRNA+molecules+using+multiple+singly+labeled+probes.">Imaging individual mRNA molecules using multiple singly labeled probes.</a> Nat. Methods. 5, 877-879 (2008).</li></ol>

C.楚和HY张被命名为基于这种方法的专利申请的发明人。

在这里，我们介绍了CHIRP-seq技术， 在体内 lncRNA的全基因组结合位点的映射方法。成功的关键参数是平铺的寡核苷酸探针和戊二醛交联的分裂池。亲和探针的设计是简单的RNA序列，无需事先的RNA的结构或功能域的知识。三个相当不同的两个物种的RNA - - 我们与roX2，TERC和热风的成功表明，啁啾seq是可能推广到许多lncRNAs。与所有的实验，护理和适当的控制是需要解释的结果。不同的的lncRNA可能需要滴定的条件，和明智变化的条件，如选择不同的亲和探针或交联剂，可突出RNA的染色质相互作用的不同方面。芯片-seq的一样，并不是所有的绑定事件是必然的功能，需要更多的研究来确定ØRNA的生物后果ccupancy的染色质。尽管如此，我们预计其他的染色相关lncRNAs的，目前在数以万计的数字8,9的研究人员的这项技术的许多有趣的应用程序。正如芯片SEQ打开门的全基因组DNA-蛋白质相互作用的探索，“RNA相互作用组”的CHIRP-SEQ研究的可能揭示生物的许多新的途径。

Buffer List:
Dissolve a pellet of complete protease inhibitor in 1 ml water as 50x stock. Make 100 mM PMSF in isopropanol (100x stock). Superase-in is used as 200x stock. Store all at -20&deg;C.
Lysis Buffer:
50 mM Tris-Cl pH 7.0 
10 mM EDTA 
1% SDS 
Always add PMSF, P.I. and Superase-in fresh before use except when washing beads
Proteinase K Buffer (for DNA)
100 mM NaCl 
10 mM TrisCl pH 8.0 (For RNA use pH 7.0) 
1 mM EDTA 
0.5% SDS 
Add 5% by volume Proteainse K (Ambion AM2546 20 mg/ml) fresh before use
Hybridization Buffer
750 mM NaCl 
1% SDS 
50 mM Tris-Cl pH 7.0 
1 mM EDTA 
15% formamide (store in the dark at 4&deg;C) 
Always add PMSF, P.I. and Superase-in fresh before use
Wash Buffer
2x NaCl and Sodium citrate (SSC) (diluted from 20x SSC Invitrogen stock) 
0.5% SDS 
Always add PMSF fresh before use
DNA elution Buffer
50 mM NaHCO3 
1% SDS
Table of specific reagents and equipment:
<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Glutaraldehyde (EM grade)</td>
			<td>Sigma Aldrich</td>
			<td>G5882-10x10ml</td>
		</tr>
		<tr>
			<td>Motorized pellet mixer</td>
			<td>VWR</td>
			<td>V8185-904</td>
		</tr>
		<tr>
			<td>Protease inhibitor</td>
			<td>Roche</td>
			<td>11873580001</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma Aldrich</td>
			<td>78830</td>
		</tr>
		<tr>
			<td>Superase-in</td>
			<td>Ambion</td>
			<td><a href="http://www.ambion.com/catalog/CatNum.php?AM2696">AM2696</a></td>
		</tr>
		<tr>
			<td>Bioruptor</td>
			<td>Diagenode</td>
			<td>UCD-200</td>
		</tr>
		<tr>
			<td>Falcon tubes (for sonication)</td>
			<td>Corning</td>
			<td>430790</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Ambion</td>
			<td>AM2546</td>
		</tr>
		<tr>
			<td>PCR purification kit</td>
			<td>Qiagen</td>
			<td>28106</td>
		</tr>
		<tr>
			<td>C-1 magnetic beads</td>
			<td>Invitrogen</td>
			<td>65002</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma Aldrich</td>
			<td>P7626-25G</td>
		</tr>
		<tr>
			<td>DynaMag-15 magnet</td>
			<td>Invitrogen</td>
			<td>123-01D</td>
		</tr>
		<tr>
			<td>DynaMag-2 magnet</td>
			<td>Invitrogen</td>
			<td>123-21D</td>
		</tr>
		<tr>
			<td>MIRNeasy mini kit</td>
			<td>Qiagen</td>
			<td>217004</td>
		</tr>
		<tr>
			<td>Rnase H</td>
			<td>Epicentre</td>
			<td>R0601K</td>
		</tr>
		<tr>
			<td>Rnase A</td>
			<td>Sigma Aldrich</td>
			<td>R4875-100MG</td>
		</tr>
		<tr>
			<td>Phase Lock Gel Heavy</td>
			<td>5 PRIME</td>
			<td>2302810</td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Invitrogen</td>
			<td>15596-018</td>
		</tr>
		<tr>
			<td>Phenol:chloroform:Isoamyl</td>
			<td>Invitrogen</td>
			<td>15593-031</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Ricca</td>
			<td>RSOC0020-1C</td>
		</tr>
		<tr>
			<td>GlycoBlue</td>
			<td>Ambion</td>
			<td>AM9515</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>J.T. Baker</td>
			<td>4057-06</td>
		</tr>
		<tr>
			<td>PBS, pH 7.4</td>
			<td>Invitrogen</td>
			<td>10010-049</td>
		</tr>
		<tr>
			<td>Elution Buffer (EB)</td>
			<td>Qiagen</td>
			<td>19086</td>
		</tr>
		<tr>
			<td>20x SSC</td>
			<td>Invitrogen</td>
			<td>15557-036</td>
		</tr>
		<tr>
			<td>10% SDS</td>
			<td>Invitrogen</td>
			<td>15553-027</td>
		</tr>
		<tr>
			<td>DNA-free</td>
			<td>Ambion</td>
			<td>AM1906</td>
		</tr>
		<tr>
			<td>Buffer kit</td>
			<td>Ambion</td>
			<td>AM9010</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Invitrogen</td>
			<td>15515-026</td>
		</tr>
	</tbody>
</table>

chromatin isolation by rna purification (chirp)

长非编码RNA的染色质状态的关键调节剂量补偿，压印和发育的基因表达1,2,3,4,5,6,7，如重要的生物过程。最近发现的成千上万的在特定的染色质修饰复合物，如多梳镇压复杂2（PRC2），的关联lnc​​RNAs与介导的组蛋白H3赖氨酸27三甲基化（H3K27me3），建议在管理中的特定基因的染色质状态的众多lncRNAs广泛的角色时尚8,9。虽然一些lncRNAs被认为在邻近基因的顺，其他lncRNAs工作的反式调节基因的远亲位于。例如， 果蝇 lncRNAs roX1和roX2绑定的雄性细胞的X染色体上的许多地区，是至关重要的剂量补偿10,11。然而，他们的结合位​​点的确切位置，不知道在高分辨率。同样，人类的lncRNA热风可以影响对H PRC2占用基因的全基因组3,12,13，但特异性是如何实现的undreds目前还不清楚。 LncRNAs也可以作为模块化脚手架，聘请多蛋白质复合物的组装。经典反义RNA支架是TERC的RNA，由于复杂的14端粒酶模板和脚手架;热风也可以作为一个PRC2脚手架和H3K4的去甲基复杂的13。 此前的研究显示映射在染色的RNA占用大量的见解15,16，但只有一次一个单一的基因位点。占用网站的大多数lncRNAs的是不知道，和染色质调控lncRNAs的角色大多已推断的lncRNA扰动的间接影响。正如染色质免疫芯片或深度测序（芯片的芯片或芯片SEQ，分别）大大改善了我们的基因组规模的蛋白质-DNA相互作用的理解，在这里我们说明了recenTLY出版策略映射在高分辨率17长的RNA入住的全基因组。基于这种方法，通过RNA纯化（CHIRP）（ 图1），染色质隔离亲和力捕捉目标lncRNA：染色质复杂，由平铺反义寡核苷酸，然后生成基因组结合位点的地图，在几百基地的决议高灵敏度和低背景。 CHIRP是适用于许多lncRNAs因为亲和探针的设计是简单的RNA序列，并要求没有RNA的结构或功能域的知识。

Watch this Scientific Journal Video about Chromatin Isolation by RNA Purification ChIRP at JoVE.com

Chromatin Isolation by RNA Purification ChIRP

CHIRP技术是一种新型快速映射约束力的长非编码RNA基因组网站（lncRNAs）。该方法采用特异性的反义平铺寡核苷酸允许枚举的lncRNA绑定基因组站点的优势。

RNA纯化的染色质隔离（CHIRP）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

chromatin-isolation-by-rna-purification-chirp

Research

JoVE Journal

Biology

Chromatin Isolation by RNA Purification (ChIRP)

85.7K 次观看.  Stanford University School of Medicine. CHIRP技术是一种新型快速映射约束力的长非编码RNA基因组网站（lncRNAs）。该方法采用特异性的反义平铺寡核苷酸允许枚举的lncRNA绑定基因组站点的优势。

视频: Chromatin Isolation by RNA Purification ChIRP

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

科研

生物学

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

分离纯化果蝇</em磁性微球的排序>外周神经元

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

改造使用含RNA寡核苷酸基因组DNA的RNA / DNA杂交一代

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

RNA含外来体的分离和鉴定

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (&tilde;100 &mu;m) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2.
SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface 3. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7.
With the exception of one report 8, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11.
In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation.

在体内肝细胞的分离纯化的肝细胞内吞其次是肝脏灌注

The liver is the metabolic center of the mammalian body and serves as a filter for the blood.  The basic architecture of the liver is illustrated in ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

kinesin的分离和纯化果蝇胚胎

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

在体外转录和Gaussia荧光素酶基因的上限由HeLa细胞转染

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

快速分离纯化线粒体移植用组织分离和鉴别过滤

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

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

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

产生和人类INO80染色质重塑复合物及其亚纯化

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

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

从小鼠胰腺RNA提取：核糖核酸酶丰富的组织

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...