由A *新加坡星撰文支持。此外，支持的A * STAR国家科学奖学金，妇女在国家科学院士和李光耀博士后奖学金1欧莱雅的MJF。里亚尔支持由NIH的ENCODE的补助金（以R01 HG004456-01和以R01 HG003521-01）。作者也承认，录像的8个像素的制作，特别是开尔文先生一生之水，张先生启翔先生和宣威甘，新加坡队，为拍摄场景，视频编辑和米歇尔张志贤女士女士西蒂·拉希姆画外音。

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没有利益冲突的声明。

CHIA-PET是开发，以确定转录调控中的长程相互作用的方法。确定的嘉PET库质量的关键因素之一是芯片材料的质量。 在视频显示的协议，采用交叉连接细胞（EGS）和甲醛的使用。交联的第二个轴承较长的间隔臂的试剂结合使用甲醛，可能有助于蛋白质不能甲醛的约束，仅3,11,15约束力。我们已经构建库，此方法已显示出强大的结合位点和长程相互作用9。然而，交联和芯片的条件应为每个感兴趣的因素优化，重要的是不要过度的交联，作为交联太多会导致碎片中的困难，通过超声，并可能导致虚假的染色质相互作用。染色质相互作用应当验证由不同的方法，如荧光原位杂交4，由嘉义PET标识。 我们建议至少100吴质材料。虽然我们已经构建从50吴质材料质量好的库，我们已经指出，允许起始原料的大量嘉PET少于16个PCR循环，从而最大限度地减少每个库的扩增和冗余库建设。这种较低的冗余与较高的独特的映射标签和高比例的可用数据，从而使染色体的测序较少的车道更全面的互动地图。最终每管装珠量应该是50μL和100μL的磁场和琼脂糖珠分别。如果包装珠体积小于说，同样预先清除空白磁或琼脂糖珠的最小包装量，以尽量减少损失的DNA-轴承珠S在随后的步骤。个子矮的小技巧大核心提示应使用移液琼脂糖珠。 继先前公布的CHIA-PET的协议被纳入下列修改。首先，磁性Ğ珠被用来在清洗，以尽量减少样品的损失。此外，我们发现非特异性条带，大小约100个​​基点和138基点，是自我结扎半连接器或/和适配器的扩增。因此，我们减少生物素的一半，连接器和454 GS20适配器，以尽量减少非特异性条带，PCR扩增过程中的浓度。接近结扎量减少，从50毫升至10毫升，在随后的纯化步骤，以尽量减少样品的损失，也节省试剂成本。我们还增加了孵化时间固定嘉PET DNA珠，链霉亲和素珠CHIA-PET的DNA，以确保最大的捕获。 在接近结扎一步，THA嵌合结扎t没有代表在体内染色质相互作用的真实，都不可避免地在一个非特定的和随机的方式产生。因此，任何CHIA-PET的实验数据的质量评估，嵌合率估计从两个不同的特定核苷酸条码TAAG和ATGT的5半交联剂的使用。 CHIA-PET的序列高通量测序后，首先来自特定结扎产品和非特异性结扎产品的链接条码组成和序列分析可以区分8。被称为嵌合体的比例（即异AB连接器）呈现在我们的内部MCF-7 RNA聚合酶II CHIA-PET的图书馆是低于15％。 嘉PET序列，其后分为两大类，即自我结扎宠物间结扎宠物。自我结扎宠物获得自我函证结扎染色质片段，而从我而得间结扎宠物NTER结扎两种不同的DNA片段。后者再细分为三个不同的类别，基于相同的染色体（染色体内间结扎宠物）或标签都被映射到两个不同的染色体（interchromosomal间结扎宠物）的基因组上的每个标签距离。我们已经开发了1 CHIA-PET的工具软件包，理清不同类别8。这将根据库中的DNA片段。一般来说，小的芯片碎片会给一个更高的分辨率，并切断这些RNA聚合酶II嘉PET库大约是4 KB。 此外，真正的染色质相互作用可以从随机噪声区分计数间结扎宠物互动集群的数量，换句话说，高PET计数集群据说有更高的概率是一个真正的染色质相互作用8 。 来过滤误报1上升，从高浓缩铀的锚定能形成随机机会间结扎宠物，统计分析框架也已制定占到任何两锚8间结扎宠物随机形成。 总之，CHIA-PET的技术允许映射在全球范围内的染色质相互作用网络。嘉义PET芯片的实施，使图书馆的复杂性和背景噪音减少。此外，芯片增加特异性染色质相互作用，使特定的染色质相互作用与特定的转录因子5的考试。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> 4-20％梯度TBE凝胶</td><td> Invitrogen公司</td><td> EC6225BOX </td><td>步骤b，6.3 </td></tr><tr><td> 5×T4 DNA与PEG连接酶缓冲液</td><td> Invitrogen公司</td><td> 46300018 </td><td>步骤B，2.2 </td></tr><tr><td> 6％TBE凝胶</td><td> Invitrogen公司</td><td> EC6263BOX </td><td>步骤b，6.8 </td></tr><tr><td>安捷伦DNA 100含量</td><td>安捷伦科技公司</td><td> 5067-1504 </td><td rowspan="3">步骤B，7.1 </td></tr><tr><td>安捷伦高灵敏度DNA检测</td><td>安捷伦科技公司</td><td> 5067-4626 </td></tr><tr><td>安捷伦生物分析仪2100 </td><td>安捷伦科技公司</td><td> G2940CA </td></tr><tr><td>离心管过滤器（自旋X） </td><td>康宁&lt;/ TD&gt; <td> CLS8160 </td><td>步骤B，3.7和6.9 </td></tr><tr><td>黑暗读者透射</td><td>克莱尔化学研究</td><td> DR46B </td><td>步骤b，6.8 </td></tr><tr><td>数字Sonifier细胞破碎</td><td>布兰森</td><td> 450D-0101063591 </td><td>步骤一，4.3 </td></tr><tr><td> DynaMag-2磁体（磁粉浓缩） </td><td> Invitrogen公司</td><td> 123-21D </td><td>步骤，7.5.1步骤B，磁珠洗涤步骤</td></tr><tr><td> DynaMag-15磁体（磁粉浓缩） </td><td> Invitrogen公司</td><td> 123.01D </td><td>步骤一，5和6 </td></tr><tr><td> DynaMag聚合酶链反应（磁粉选矿厂） </td><td> Invitrogen公司</td><td> 49-2025 </td><td>步骤b，6.5 </td></tr><tr><td> 大肠杆菌 DNA聚合酶I </td><td> NEB </td><td> M0209 </td><td>步骤b，5.6 </td></tr><tr><td> GlycoBlue </td><td> Ambion公司</td><td> AM9516 </TD&gt; <td>步骤一，7.5.1步骤B，4.3和6.5 </td></tr><tr><td> Illumina的CBOT集群发电系统</td><td> Illumina公司</td><td> SY-301-2002 </td><td>步骤B，7.4 </td></tr><tr><td> Illumina的基因组分析仪IIx能</td><td> Illumina公司</td><td> SY-301-1301 </td><td>步骤B，7.5 </td></tr><tr><td> Illumina的体育引子</td><td> Illumina公司</td><td> PE-102-1004 </td><td>步骤B，5.4（如有必要） </td></tr><tr><td>智能混合机</td><td>百利高技</td><td> RM-2L </td><td>步骤b，任何孵化与旋转</td></tr><tr><td>的LightCycler 480实时PCR系统</td><td>罗氏公司</td><td> 04 640 268 001 </td><td>步骤一，7.5.3步骤B，7.3 </td></tr><tr><td> LightCycler480 DNA SYBR Green I的MasterMix </td><td>罗氏公司</td><td> 03 752 186 001 </td><td>步骤B，7.5.3 </td></tr><tr><td>的M-280链霉亲和素磁珠</td><td> Invitrogen公司</td><td> 11206D </td><td>步骤b，5.2 </td></tr> &lt;TR&gt; <td>磁（磁珠）G蛋白</td><td> Invitrogen公司</td><td> 100.03D </td><td>步骤一，5.1.1和5.2.1 </td></tr><tr><td> MaXtract高密度（2毫升） </td><td> Qiagen公司</td><td> 129056 </td><td>步骤一，7.5.1 </td></tr><tr><td> MaXtract高密度（50毫升） </td><td> Qiagen公司</td><td> 129073 </td><td>步骤B，4.2 </td></tr><tr><td> MmeI </td><td> NEB </td><td> R0637 </td><td> R0637 </td></tr><tr><td>核糖核酸酶一核糖核酸</td><td> Promega公司</td><td> M426C </td><td>步骤B，4.8 </td></tr><tr><td> RNA聚合酶II（8WG16）单克隆抗体</td><td> Covance公司</td><td> MMS-126R </td><td>步骤一，5.2.4 </td></tr><tr><td>橡树岭离心管（聚丙烯） </td><td> NALGENE </td><td> 3119-0050 </td><td>步骤一，3.1 </td></tr><tr><td>橡树岭离心管（铁氟龙FEP） </td><td> NALGENE </td><td> 3114-0050 </td><td>步骤B，4.3 </td></tr><tr><td> phus离子高保真预混</td><td> finnzymes </td><td>架F-531 </td><td>步骤b，6 </td></tr><tr><td> picogreen（QUANT-IT）双链DNA试剂</td><td> Invitrogen公司</td><td> P11495 </td><td>步骤一，7.5.2 </td></tr><tr><td>聚苯乙烯圆底试管</td><td> BD公司</td><td> 352057 </td><td>步骤一，4.1 </td></tr><tr><td>蛋白酶抑制剂片（完整，EDTA免费） </td><td>罗氏公司</td><td> 11873580001 </td><td>步骤一，1.6起</td></tr><tr><td>蛋白酶K溶液（20mg/mL时） </td><td> Fermentas公司</td><td> E00491 </td><td>步骤A，4.4，7.5.1 步骤B，4.1 </td></tr><tr><td> T4 DNA连接酶</td><td> Fermentas公司</td><td> EL0013 </td><td>步骤B，2.2，3.9和5.4 </td></tr><tr><td> T4 DNA连接酶缓冲液（NEB） </td><td> NEB </td><td> B0202S </td><td>步骤B，3.3和3.9 </td></tr><tr><td> T4 DNA聚合酶</td><td> Promega公司</td><td> M4215 </td><td>步骤B，1.2 </td></tr><tr><td> T4 DNA多聚核苷酸激酶</td><td> NEB </td><td> M0201 </td><td>步骤b，3.3 </td></tr><tr><td> TruSeq SBS的工具包V5-GA </td><td> Illumina公司</td><td> FC-104-5001 </td><td>高中会考的Illumina的Genome Analyzer的IIx能系统</td></tr><tr><td> TruSeq体育群集工具包V2-CBOT-GA </td><td> Illumina公司</td><td> PE-300-2001 </td><td>利用Illumina的CBOT集群发电系统</td></tr><tr><td> SYBR Green I的</td><td> Invitrogen公司</td><td>的S-7585 </td><td>步骤B，6.3和6.8 </td></tr><tr><td> Xcell的的SureLock迷你细胞电泳系统</td><td> Invitrogen公司</td><td> EI0001 </td><td>步骤B，6.3和6.8 </td></tr></tbody></table><table border="1"><tbody><tr><td> 名称 </td><td colspan="2"> 序列 </td><td> 评论 </td></tr><tr><td rowspan="2">生物素的一半，连接器（200ng/μl） </td><td>顶部</td><td> 5&#39;GG化学计算海巡署/ iBiodT / ATC TTA台泥的AAC 3&#39; </td><td> 250 nmole的规模高效液相色谱纯化内部生物素DT（9） </td></tr><tr><td>博特</td><td> 5&#39;滋养细胞的TAA GGA GAT的空中交通管制气相色谱3&#39; </td><td> 250 nmole的规模高效液相色谱纯化</td></tr><tr><td rowspan="2">生物素的一半，连接器（200ng/μl） </td><td>顶部</td><td> 5&#39;GGC政府总部大楼的GA / iBiodT / ATA CAT台泥的AAC 3&#39; </td><td> 250 nmole的规模高效液相色谱纯化内部生物素DT（9） </td></tr><tr><td>博特</td><td>糖耐量GGA ATG的5&#39;达航管气相色谱3&#39; </td><td> 250 nmole的规模高效液相色谱纯化</td></tr><tr><td rowspan="2">非生物素半交联剂（200ng/μl） </td><td>顶部</td><td> 5&#39;GGC GAT的政府总部大楼航管GGA台泥的AAC 3&#39; </td><td> 250 nmole的规模PCR级</td></tr><tr><td>博特</td><td>糖耐量GGA TCC的5&#39;GAT的空中交通管制的GC 3&#39; </td><td> 250 nmole的规模PCR级</td></tr><tr><td rowspan="2"> GS20适配器一个（200ng/μl） </td><td>顶部</td><td>文建大老山隧道的CAT 5&#39;CCC认证炉TGC GTG台泥的CATCTG的交委会建CCC认证TGT的反恐活动星系核的N 3“ </td><td> 250 nmole的规模PCR级</td></tr><tr><td>博特</td><td> AGA公司5&#39;CTG造影GGA GGG的AAC AGA公司TGG GAC的ACG的造影GGA热重的GAT的GG 3&#39; </td><td> 250 nmole的规模PCR级</td></tr><tr><td rowspan="2"> GS20适配器乙（200ng/μl） </td><td>顶部</td><td> 5&#39;CTG的AGA公司成飞贲门癌ACA的GGG GAT的AGG的民航局GGC ACA的造影GGG的ATA的GG 3&#39; </td><td> 250 nmole的规模PCR级</td></tr><tr><td>博特</td><td> 5&#39;建航管CCC认证的TGT GTG建炉TGC税务师台泥建糖耐量GCG TGT的反恐活动星系核的N 3“ </td><td> 250 nmole的规模PCR级</td></tr><tr><td rowspan="2"> Illumina公司的神经网络适配器</td><td>顶部</td><td> 5&#39;ACA的反恐TTT CCC认证交咨会ACG的ACG的反恐交委会海巡署TC 3&#39; </td><td> 250 nmole的规模高效液相色谱纯化步骤B，5.4 </td></tr><tr><td>博特</td><td> 5&#39;磷酸 - CGG公司的GAT AAG的AGC的GGT的TCA的GCA的GGA ATG的化学计算公司3&#39; </td><td> 250 nmole的规模高效液相色谱法净化磷酸的5&#39;端，请参阅步骤B，5.4 </td></tr><tr><td> Illumina的1-454（前锋）引物（10μM） </td><td colspan="2"> 5，亚洲空运中心的GAT ACG的图文影像集团行政协调行政协调GAG的空中交通管制交咨会行政协调会税务师台泥建GTG炉TGC CTT的G 1“ </td><td> 250 nmole的规模PCR级</td></tr><tr><td> Illumina的2-454（反向）引物（10μM） </td><td colspan="2"> 5&#39;GCA的棉酚民航局海关总署GGC ATA的海巡署的GAT CGG公司台泥空管TCA的台泥CTG的总工会GTC的3&#39; </td><td> 250 nmole的规模PCR级</td></tr><tr><td> 1.1定量PCR引物（10μM） </td><td colspan="2"> 5&#39;亚洲空运中心的GAT ACG的图文影像集团行政协调行政协调聚糖3&#39; </td><td> 10nmole规模PCR级</td></tr><tr><td> 2.1定量PCR引物（10μM） </td><td colspan="2">民航局贲门癌棉酚海关总署5&#39;GGC ATA的海巡署3&#39; </td><td> 10nmole规模PCR级</td></tr><tr><td> 3-454 Illumina的测序引物（100μM） </td><td colspan="2"> 5&#39;TGC GTG TCC的CAT CTG的交委会建CCC认证的TGT反恐公司3&#39; </td><td> 100nmole规模高效液相色谱纯化</td></tr><tr><td> 4-454 Illumina的测序引物（100μM） </td><td colspan="2"> 5&#39;GTG建TGC税务师台泥建糖耐量图文影像集团的TGT反恐公司3&#39; </td><td> 100nmole规模高效液相色谱纯化</td></tr></tbody></table> 寡核苷酸和适配器表。综合DNA技术（IDT）的连接器和适配器，并准备在以同样的方式订购寡核苷酸前面所述10。半连接器和适配器，可事先准备和存放于-20℃的几个月

chromatin interaction analysis with paired-end tag sequencing (chia-pet) for mapping chromatin interactions and understanding transcription regulation

基因组被组织成三维结构，采用高阶构内，2，12微米大小的核空间。这样的架构是不是随机的，涉及13基因启动子调控元件之间的相互作用。具体的调控序列的转录因子结合，带来了网络的转录调控和协调1，14。 配对末端标签测序（嘉PET）的染色质相互作用分析，以确定这些高阶染色质结构5,6。细胞是固定的，相互作用的位点是由共价DNA-蛋白质交联抓获。为了尽量减少非特异性噪声和降低复杂性，以及增加特异性染色质相互作用分析，染色质免疫沉淀（ChIP）是用来对付特定的蛋白质因子丰富接近结扎前的染色体片段的利益。随后结扎涉及半交联剂对一起拴在个别染色质复合物的DNA片段之间形成的共价链接。的侧翼MmeI，限制在一半交联剂酶切位点，允许提取配对结束后MmeI消化标记链接标记构造（PETS）。作为交联剂一半是生物素，这些PET结构采用链霉亲和磁珠纯化。纯化的宠物结扎与新一代测序适配器和通过下一代，如Illumina的Genome Analyzer的时序产生相互作用的片段目录。映射和生物信息学分析，然后进行识别芯片浓缩铀的结合位点和芯片丰富的染色质相互作用8。 我们已经制作了一个视频演示，CHIA-PET的协议，特别是芯片制备的关键方面，由于芯片的质量起到了重要作用，在的嘉PET库结果。由于协议很长的，唯一的关键步骤是在视频显示。

Watch this Scientific Journal Video about Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation at JoVE.c

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

配对末端标签测序（嘉PET）的染色质相互作用分析方法是<em&gt;从头</em&gt;染色质相互作用的检测，为更好地理解转录调控。

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 ...

chromatin-interaction-analysis-with-paired-end-tag-sequencing-chia

Research

JoVE Journal

Biology

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

30.5K 次观看.  Agency for Science, Technology and Research, Singapore. 配对末端标签测序（嘉PET）的染色质相互作用分析方法是从头染色质相互作用的检测，为更好地理解转录调控。

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

A Chromatin Assay for Human Brain Tissue

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental factors that might result in epigenetic alterations of gene expression and other molecular pathology. Traditionally, however, expression studies in postmortem brain were confined to quantification of mRNA or protein. The limitations encountered in postmortem brain research   such as variabilities in autolysis time and tissue integrities   are also likely to impact any studies of higher order chromatin structures. However, the nucleosomal organization of genomic DNA   including DNA:core histone binding - appears to be largely preserved in representative samples provided by various brain banks. Therefore, it is possible to study the methylation pattern and other covalent modifications of the core histones at defined genomic loci in postmortem brain. Here, we present a simplified native chromatin immunoprecipitation (NChIP) protocol for frozen (never-fixed) human brain specimens. Starting with micrococcal nuclease digestion of brain homogenates, NChIP followed by qPCR can be completed within three days. The methodology presented here should be useful to elucidate epigenetic mechanisms of gene expression in normal and diseased human brain.

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

一个人脑组织中的染色质含量

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

科研

生物学

Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other genome-wide analyses. mRNA-Seq even opens the gate for gene expression analysis of non-sequenced genomes. mRNA-Seq offers high sensitivity, a large dynamic range and allows measurement of transcript copy numbers in a sample. Illumina&#x2019;s genome analyzer performs sequencing of a large number (&gt; 107) of relatively short sequence reads (&lt; 150 bp).The &quot;paired end&quot; approach, wherein a single long read is sequenced at both its ends, allows for tracking alternate splice junctions, insertions and deletions, and is useful for de novo transcriptome assembly.
One of the major challenges faced by researchers is a limited amount of starting material. For example, in experiments where cells are harvested by laser micro-dissection, available starting total RNA may measure in nanograms. Preparation of mRNA-Seq libraries from such samples have been described1, 2 but involves significant PCR amplification that may introduce bias. Other RNA-Seq library construction procedures with minimal PCR amplification have been published3, 4 but require microgram amounts of starting total RNA.
Here we describe a protocol for the Illumina Genome Analyzer II platform for mRNA-Seq sequencing for library preparation that avoids significant PCR amplification and requires only 10 nanograms of total RNA. While this protocol has been described previously and validated for single-end sequencing5, where it was shown to produce directional libraries without introducing significant amplification bias, here we validate it further for use as a paired end protocol. We selectively amplify polyadenylated messenger RNAs from starting total RNA using the T7 based Eberwine linear amplification method, coined &quot;T7LA&quot; (T7 linear amplification). The amplified poly-A mRNAs are fragmented, reverse transcribed and adapter ligated to produce the final sequencing library. For both single read and paired end runs, sequences are mapped to the human transcriptome6 and normalized so that data from multiple runs can be compared. We report the gene expression measurement in units of transcripts per million (TPM), which is a superior measure to RPKM when comparing samples7.

Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.

单次读取和配对的最终从10微克总RNA的mRNA - Seq的Illumina的图书馆

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

染色质免疫沉淀（ChIP）使用果蝇组织

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

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 ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

代高通量测序技术（ChIP-seq的高品质的染色质免疫沉淀DNA模板）

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

定量，单细胞在全安装胚珠在染色质修饰和核体系结构分析的有效方法<em&gt;拟南芥</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

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 ...

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

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

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

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

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 ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

具体的基因组区域及相关分子鉴定enChIP隔离

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

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).