Name: high-throughput identification of gene regulatory sequences using next-generation sequencing of circular chromosome conformation capture (4c-seq)
Uploaded: 2018-10-05T13:00:00
Description: Watch this Scientific Journal Video about High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq at JoVE.com

这项工作得到了结缔组织国立研究院 (R01AR065523) 的支持。

1. 限制酶的选择
<ol><li>确定一个感兴趣的区域 (例如, 基因启动子, 单核苷酸多态性 (SNP), 增强剂), 并从资料库中获取 DNA 序列, 如国家生物技术信息中心 (NCBI)。</li>
	<li>确定第一限制酶消化 (RE1) 中的候选限制酶 (REs), 它不在感兴趣的序列内切割, 在重新消化后产生粘性端 (DNA 终点), 其活动不受中央甲基化。 注意: 数据的分辨率由 RE1 决定。一个6基对 (bp) 识别序列的酶平均产生大约 4 kilobases...

<ol>
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4C 结果有可能揭示染色质相互作用, 可以识别以前未知的调控元素和/或目标基因是重要的在特定的生物环境24,25,26。然而, 技术障碍可能会限制从这些实验获得的数据。在4C 协议中, 模板过度放大引起的 PCR 偏倚很可能。该协议通过利用 qPCR 来确定最佳的放大周期数以客观的方式来解决这个问题。此外, 通过限制文摘从扩增的反?...

DNA 元素百科全书 (编码) 项目为人类基因组1、280% 的功能活动提供了综合注解, 从而促进了基因表达的调控元素的识别。在体内转录因子结合, DNaseI 过敏, 和表观组蛋白和 DNA 甲基化修饰的个体细胞类型的地点的鉴定为候选调控的功能分析铺平了道路。目标基因表达的元素。有了这些发现, 我们面临着确定调控元素和基因之间功能互联的挑战。具体来说, 一个给定的目标基因和它的增强剂之间的关系是什么？染色质构象捕获 ( 3C ) 方法通过在固定染色质 3 中通过捕获的事件识别一个感兴趣区域和候选交互序列之间的物理和可能的功能互 , 直接解决这个问题 ..然而, 随着我们对染色质相互作用的理解增加, 显然, 对预选候选基因座的调查不足以提供对基因增强器相互作用的完全理解。例如, 编码使用高通量染色体构象捕获碳拷贝 (5C) 方法来检查人类基因组的一小部分 (1%, 44 个基因座的先导集), 并报告了基因座的复杂互联。基因和促进剂与确定的相互作用平均2–4不同的相互作用的伙伴, 其中许多是数以百计的 kilobases 离开在线性空间4。还有, 李等。采用配对端标记测序 (嘉-PET) 进行染色质相互作用分析, 分析全基因组启动子相互作用, 发现65% 的 RNA 聚合酶 II 结合部位参与了染色质的相互作用。其中一些相互作用导致大, 多基因复合体横跨数以百计的 kilobases 基因组距离和包含, 平均, 8-9 个基因每5。这些发现结合在一起, 强调需要无偏全基因组方法来审问染色质相互作用。其中一些方法在施密特和al中进行了评述。6。
最近的染色质构象捕获研究方法, 加上下一代测序 (高 c 和 4 C 序列), 使发现未知数列与感兴趣的区域6。具体来说, 用下一代测序 (4 c 序列) 建立环状染色体构象捕获, 以以无偏见的方式7通过测序 DNA 从近距离捕获的染色质到3D 空间感兴趣的区域。简要地说, 染色质是固定的, 以保持其原生蛋白-DNA 相互作用, 与限制酶, 并随后结扎在稀释条件下, 以捕获生物学相关的 "缠结" 的相互作用的基因座 (图 1)。交叉链接被逆转, 以去除蛋白质, 从而留下的 DNA 可用于额外的分裂与第二限制酶。最后的结扎会产生更小的相互作用的圆圈。然后用引物对感兴趣的序列进行生成, 从发送的片段中产生未知序列的放大库, 其次是下游下一代测序。
此处提出的关于样本准备的协议对现有的4种 c 序列方法8、9、10、11、12进行了两项重大修改。首先, 它使用一种基于 qPCR 的方法来经验主义地确定 4 c 序列库准备步骤的最佳放大周期数, 从而减少了由于图书馆过度扩增而导致的 PCR 偏倚的可能性。其次, 它使用额外的限制摘要步骤, 努力减少已知的 "诱饵" 序列的一致性, 这会阻碍测序仪精确地进行基调用, 从而使每读中的唯一信息序列最大化。其他协议通过将许多 (12-15)8 4 c 序列库与不同的诱饵顺序和/或限制站点汇集在一起来规避这个问题, 这是其他实验室可能无法实现的大量实验。此处所做的修改允许少量的实验、样本和/或复制被编入索引, 并汇集到一个单独的车道中。

<table>
	<tbody>
		<tr>
			<td>HindIII</td>
			<td>NEB</td>
			<td>R0104S</td>
			<td></td>
		</tr>
		<tr>
			<td>CviQI</td>
			<td>NEB</td>
			<td>R0639S</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA oligonucleotide primers</td>
			<td>IDT</td>
			<td></td>
			<td>To be designed by the reader</td>
		</tr>
		<tr>
			<td>50 mL conical centrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>06-443-19</td>
			<td></td>
		</tr>
		<tr>
			<td>1.7 mL microcentrifuge tubes</td>
			<td>MidSci</td>
			<td>AVSS1700</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Thermo Fisher</td>
			<td>14190-136</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde, methanol free</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutator</td>
			<td>VWR</td>
			<td>15172-203</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>JT Baker</td>
			<td>4059-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated microcentrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>ED2SS</td>
			<td></td>
		</tr>
		<tr>
			<td>20% SDS solution</td>
			<td>Sigma-Aldrich</td>
			<td>05030</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Thermo Fisher</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-143</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slips</td>
			<td>VWR</td>
			<td>16004-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Alfa Aesar</td>
			<td>A16046</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking heat block</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2M Tris-HCl</td>
			<td>Quality Biological</td>
			<td>351-048-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>NEB</td>
			<td>P8107S</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol:chloroform:isoamyl alcohol (25:24:1)</td>
			<td>Sigma-Aldrich</td>
			<td>P2069</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>M5661</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mg/mL glycogen</td>
			<td>Thermo Fisher</td>
			<td>R0561</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>04-355-223</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>Fisher Scientific</td>
			<td>MT-46-000-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>qPCR cycler</td>
			<td>Thermo Fisher</td>
			<td>4453536</td>
			<td></td>
		</tr>
		<tr>
			<td>qPCR plates</td>
			<td>Thermo Fisher</td>
			<td>4309849</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>Thermo Fisher</td>
			<td>4375786</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR strip tubes</td>
			<td>MidSci</td>
			<td>AVSST-FL</td>
			<td></td>
		</tr>
		<tr>
			<td>1M Magnesium chloride</td>
			<td>Quality Biological</td>
			<td>351-033-721</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreotol</td>
			<td>Sigma-Aldrich</td>
			<td>43815</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine triphosphate</td>
			<td>Sigma-Aldrich</td>
			<td>A2383</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>NEB</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A6013</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase A</td>
			<td>Thermo Fisher</td>
			<td>EN0531</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiaquick PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr>
			<td>MinElute PCR Purification kit</td>
			<td>Qiagen</td>
			<td>28004</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Expand Long Template PCR System</td>
			<td>Sigma-Aldrich</td>
			<td>11681834001</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP mix</td>
			<td>Thermo Fisher</td>
			<td>R0191</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Green I</td>
			<td>Sigma-Aldrich</td>
			<td>S9430</td>
			<td></td>
		</tr>
		<tr>
			<td>ROX</td>
			<td>BioRad</td>
			<td>172-5858</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>End-It DNA End-Repair Kit</td>
			<td>Lucigen</td>
			<td>ER0720</td>
			<td></td>
		</tr>
		<tr>
			<td>LigaFast Rapid DNA Ligation System</td>
			<td>Promega</td>
			<td>M8221</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Safe</td>
			<td>Thermo Fisher</td>
			<td>S33102</td>
			<td></td>
		</tr>
		<tr>
			<td>Taq Polymerase</td>
			<td>NEB</td>
			<td>M0267S</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraSieve Agarose</td>
			<td>IBI Scientific</td>
			<td>IB70054</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiaquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput identification of gene regulatory sequences using next-generation sequencing of circular chromosome conformation capture (4c-seq)

由于调控元素对目标基因的定位和作用大小的变化, 确定特定目标基因的调控元素构成了重大的技术挑战。通过保守转录因子结合位点的生物信息学预测与活化基因表达相关的近表观遗传修饰的存在和功能, 取得了一些进展。染色质构象捕获研究已经改变了我们的能力, 发现在序列之间的物理染色质接触, 甚至在整个基因组。环形染色质构象捕获与下一代测序 (4 c 序列), 特别是设计, 以发现所有可能的物理染色质相互作用的特定序列的利益 (观点), 如目标基因或监管增强。目前的 4 c 序列策略直接从视点内排列, 但需要多个不同的视点同时进行排序, 以避免与下一代测序平台一致的基调用 (成像) 技术难题。这一量的实验对许多实验室来说可能并不实际。在这里, 我们报告了一个改进的方法, 4 c 序列协议, 包括了额外的限制酶文摘和基于 qPCR 的放大步骤, 旨在促进更大的捕获不同的序列读取和减少可能的PCR 偏倚, 分别。我们修改后的4C 方法适合于标准分子生物学实验室评估染色质体系结构。

从2–3丢弃的新生儿包皮、池中和培养的 KSFM 中分离出主要的人角质形成细胞, 辅以30µg/毫升牛垂体提取物、0.26 毫升重组人表皮生长因子和0.09 毫米氯化钙 (CaCl2) 摄氏37摄氏度, 5% 二氧化碳。这些细胞被分成两个烧瓶, 一个烧瓶由添加 CaCl2到最终浓度为1.2 毫米的72小时 107细胞分别被固定在1%室温下甲醛为10分钟。另外, K562 细胞生长在 RPMI 补充10% 胎牛?...

Watch this Scientific Journal Video about High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq at JoVE.com

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

基因和调控元素之间的物理相互作用的识别是具有挑战性的, 但已被染色体构象捕获方法所促进。这种对 4 c 序列协议的修改可以减少 pcr 模板的过度放大, 并通过加入限制酶消化步骤来最大限度地提高 mappability 的读数。

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

此方法可以回答转录调控领域中的关键问题，例如染色质相互作用的作用。这种技术的主要优点是，它为感兴趣的一个中心提供了相对公正的交互捕获。虽然此方法可以提供对促进剂增强剂相互作用的洞察，但它也可以用于识别其他类型的染色质相互作用。通常，对此方法的个体将难以操作，因为协议需要几天时间，并且底法设计需要试验和错误以及非典型底法方向。为了保持选择细胞中的染色质相互作用，通过每1000万细胞在PBS中加入9.5毫升1%电子显微镜级甲醛来交叉链接它们。在室温下摇动10分钟时孵育细胞。将反应管转移到冰中，以淬火交汇反应，将冰冷一摩尔甘油加入0.125摩尔的最终浓度，然后轻轻反转混合。在根据文本协议离心和清洗细胞后，用0.5%SDS和一种X蛋白酶抑制剂将颗粒悬浮在5毫摩尔EDTA的125微升中。在冰上孵育细胞悬浮液10分钟。为确保细胞 lyse 完整，在显微镜幻灯片上将六个微升的细胞与六个微升的 trypan 蓝色混合，并盖上盖板。在显微镜下查看，查看被解的细胞内部为蓝色，未解白细胞为白色。对于第一个限制消化添加30微升10X限制酶缓冲液，和27微升20%Triton X-100到细胞悬浮液，并调整总体积到300微升与水。拆下 15 微升等分，在 4 摄氏度下存储为未消化控制。在剩余的反应混合物中加入200个约束酶的一体。在900 RPM搅拌的震动加热块中，在适合酶的温度下孵育过夜。第二天，再加入200个单位的酶，并持续一夜的这种孵化。取出 15 微升等分，储存在 4 摄氏度下作为消化控制。为了确定消化效率，在每个未消化和消化的对照装置中添加 82.5 微升 10 毫摩尔 Tris-HCl，pH 7.5。加入2.5微升的蛋白酶K，在65摄氏度下孵育一小时，以逆转甲醛交叉连接。要分离消化的DNA，在管中加入100微升酚氯仿。通过几个快速反转混合，去除残留的蛋白质污染。在室温下在 16，100 G 下离心 5 分钟。离心后，将水相转移到新管中。将醋酸钠、糖原和100%乙醇加入管中，通过反转轻轻混合。在零下80摄氏度下孵育一小时，以沉淀DNA。在摄氏4度的16，100 G下将管离心20分钟。取出上一液，然后加入500微升70%乙醇洗涤DNA颗粒。在室温下在 16，100 G 下离心 5 分钟。去除上一杯空气后，在室温下干燥颗粒两分钟，以去除残留乙醇。将干颗粒重新悬浮在50微升无核酸酶无核酸水中，然后按照文本协议中所述，通过QPCR确定消化效率。为了准备结扎，加热和激活限制酶，在65摄氏度下孵育管子20分钟。然后将管内的内容转移到50毫升锥形管中，加入无核酸酶、10 X连体缓冲液和T4 DNA连结酶。通过旋转轻轻混合，在16摄氏度下孵育过夜，然后按照文本协议中所述进行。反向交叉链接添加15微升蛋白酶K，并在65摄氏度过夜孵育。第二天，加入30微升的RNase A，并在37摄氏度下45分钟孵育。要继续进行染色质分离，加入七毫升苯酚氯仿，通过几个快速反转混合。在室温下将管在3，300 G下离心15分钟。离心后，将水相转移到新的50毫升管中，加入无核酸酶、三摩尔醋酸钠、糖原和100%乙醇。混合内容物，在负80摄氏度下孵育一小时。孵育后，在4摄氏度的3，900G下将管离心20分钟。取出上清液，然后用10毫升冰70%乙醇清洗颗粒。在 3，300 G 的 4 摄氏度下离心 15 分钟。取出上一液，并在室温下短暂干燥颗粒。在 37 摄氏度下将颗粒溶解在 150 微升中，10 毫摩尔三升-HCl pH 7.5。储存在负20摄氏度或继续第二次限制消化，结扎和DNA纯化，如文本协议中所述。DNA纯化后，使用反向PCR底因剂和网络及ROX染料执行QPCR，以确定对未知相互作用序列进行反向PCR放大的放大周期数，如本文所述。为了准备DNA的测序，通过消化一微克的纯化产品反向PCR以及限制性酶监测器，修剪第三个限制消化的诱饵序列。在与预期片段的浓度的凝固糖凝胶上运行消化的限制性酶或 RE 监测器。一旦消化效率由凝胶电泳确定，继续反向PCR产品纯化和测序库的准备，如文本协议中所述。第三次限制消化对于下一代循环染色体确认捕获测序方案非常重要。这种消化修剪诱饵序列，这可以帮助确定染色质的吸引力。已消化的 RE 监测仪的 Agarose 凝胶电泳表明，对并行消化的反向 PCR 产品有充分的消化。完成四个C测序读取被修剪并映射到人类参考基因组hg38，使用BWA软件包。大多数读取在限制站点印地语III 或 CviQ1I 站点与印地语网站相邻，如预期的那样对齐。在尝试此过程时，确保细胞被lysed和染色质被充分消化是重要的。按照此过程，可以执行其他方法，如染色质免疫沉淀或 CHIP，以回答其他问题，如识别染色质相互作用中涉及的转录因子。该技术开发后，为染色质领域的研究人员探索物理调控网络铺平了道路。

Research

JoVE Journal

Genetics

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

适用于下一代测序的植物细菌DNA富集方法的优化与比较分析

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic ...

科研

遗传学

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

核密度分析的天然染色质沉淀测序文库的生成

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

通过重叠2方法高通量识别协同药物组合

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease

有针对性的下一代测序和生物信息学管道评价宪政疾病的遗传决定因素

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The ...

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

用于下一代测序的近全长 HIV-1 Proviruses 的扩增

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

Identification of Circular RNAs using RNA Sequencing

使用RNA测序识别循环RNA

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein ...

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing (ChIP-seq)

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing ChIP-seq

使用染色质免疫沉淀和高通量测序（ChIP-seq）发现沙门氏菌生物膜中的CsgD监管目标

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a technique that can be used to discover the regulatory targets of transcription ...

Identification of Enhancer-Promoter Contacts in Embryoid Bodies by Quantitative Chromosome Conformation Capture (4C)

Identification of Enhancer-Promoter Contacts in Embryoid Bodies by Quantitative Chromosome Conformation Capture 4C

通过定量染色体形成捕获（4C）鉴定胚胎体增强剂-促进者接触

During mammalian development, cell fates are determined through the establishment of regulatory networks that define the specificity, timing, and spatial ...

Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter

使用下一代测序来鉴定与CD4启动子附近CAS9诱导的双链断裂修复相关的突变

Double strand breaks (DSBs) in DNA are the most cytotoxic type of DNA damage. Because a myriad of insults can result in these lesions (e.g., replication ...

Pre-Implantation Genetic Testing for Aneuploidy on a Semiconductor Based Next-Generation Sequencing Platform

基于半导体的下一代测序平台上非整倍体的植入前基因检测

Next-generation sequencing has gained increasing importance in the clinical application in the determination of genetic variants. In the pre-implantation ...