这项工作是支持学院发展奖由来自费城儿童医院。

1。跨链接染色的<ol><li> 100x20mm细胞培养皿中的细胞生长。细胞的量的范围可以从1到10百万个细胞每皿取决于细胞的类型。约2万个细胞就足够一个免疫沉淀。 </li><li>交叉链接的细胞偶尔晃动10分钟，在室温下，用1％的甲醛。 </li><li>淬火通过加入终浓度为125 mM的甘氨酸与孵育5分钟，在室温下交联。 </li><li>洗涤细胞用1X磷酸盐缓冲盐水（PBS）两次，倾析PBS中，然后加入0.2毫升的PBS。 </li><li>收集细胞用塑料刮板细胞的成离心管。 </li><li>自旋向下的细胞在4℃下以2,000 rpm 5分钟</li><li>吸出上清液。 SDS裂解缓冲液中重悬细胞（1％SDS，10mM的乙二胺四乙酸（EDTA）的50mM Tris-HCl pH值8.1）的全细胞裂解物，或将其作为一个颗粒的核提取。 </li><li>细胞可以被保存在-80℃或一个可以请立即进行伊利与超声。 </li></ol> 2。准备核（继续执行步骤3.5全细胞裂解液） <ol><li>用1X蛋白酶抑制剂的每个实验中，补充细胞裂解缓冲液（5mM的筒pH为8.0，85 mM的氯化钾，0.5％NP-40）。 </li><li>解冻细胞沉淀重悬细胞裂解液颗粒体积约10倍。 </li><li> DOUNCE均化10倍杵，然后在冰上孵育10分钟。 </li><li>在4000转离心样品在4℃5分钟，弃上清，保存核颗粒。 </li></ol> 3。超声* <ol><li>热身SDS裂解缓冲液和使用蛋白酶抑制剂的缓冲液补充量。 </li><li>在SDS裂解缓冲液悬浮核颗粒（约0.5毫升的缓冲液，每1-10万细胞） </li><li>在冰上孵育10分钟。 </li><li>添加0.5毫升离心管样品。 </li><li> 30秒和45秒使用Misonix的超声波仪，超声湿冰关闭幅度设置为2。可以首先通过尝试不同的循环数（例如，2，4，8，12，16，和20个或更多个周期）来确定理想的片段大小的周期数。可用于不同品牌的超声波破碎，然而，情况会有所不同。实验与数量的周期上的时间和关闭必须进行，以便确定了理想的条件。 </li><li>收集超声检查结果，并做定量分析，每个样品20微升。可以储存在-80℃下的样品的其余部分</li><li>通过加入30微升的0.1×TE缓冲液稀释20微升的样品。 </li><li>治疗核糖核酸酶1μl的样品在37℃，1小时，2小时，然后加入1μl蛋白酶K和孵化，在62°C。 </li><li>运行20μl的样品在2％琼脂糖凝胶上。 </li><li>然后用QIAquick PCR纯化试剂盒纯化样品的剩余量量化使用的NanoDrop分光光度计。 </li></ol> *本地驰P，microccocal核酸酶消化交替使用剪切的DNA。 4。座琼脂糖珠* <ol><li>如果已经堵塞珠，继续步骤5.1。 </li><li>使用蛋白A或蛋白G琼脂糖。对于5免疫沉淀（IPS），用600微升50％珠浆（300微升珠颗粒） </li><li>洗了有孔玻璃珠，以800rpm旋转，在4℃，持续1分钟，弃去上清液。加入略超过2毫升的ChIP稀释缓冲液（0.01％SDS，1.2 mM的EDTA，167 mM氯化钠，1.1％的Triton X-100，16.7毫米的Tris-HCl pH为8.1），并混合通过缓慢颠倒试管10X。再次停止旋转，在800rpm，在4℃下1分钟，弃去上清液。重复洗涤2次以上。 </li><li>座有孔玻璃珠，用封闭液在4℃过夜旋转。封闭液的配方参考表1。 </li></ol> 5。预清除染色<ol><li>超声处理染色质在冰上解冻。 </li><li>降速12000转f或10分钟，在4°C，然后把冰马上删除SDS（白色颗粒）。 </li><li>收集上清液，弃去沉淀，如果有必要，并结合样品。 </li><li>取出实验所需金额的基础上计算（1-10微克每IP的染色质）。 </li><li>稀质10X芯片的稀释液，辅以蛋白酶抑制剂。 </li><li>加入100μl每个IP封锁珠。 </li><li>在4℃旋转1小时。 </li></ol> 6。免疫沉淀<ol><li>停止旋转，样品在800 rpm离心1分钟，将上清转移至新管。 </li><li>降速上清液在800转1分钟，转移到另一个干净的试管。 </li><li>保存20微升上层清液，作为输入控制，在-20°C </li><li>等分试样的染色质在实验中进行的IP地址的数量。 </li><li>新增2微克抗体染色质每1-10微克到每个样品。 </li><li>孵育过夜4℃旋转。 </li><li>加入100μl的封锁珠每个IP样本。 </li><li>孵育1小时，在4℃下旋转。 </li><li>颗粒的有孔玻璃珠通过降低盘片转速在800rpm，持续1分钟，并丢弃尽可能多的上清液。 </li><li>一次低盐免疫复合物洗涤缓冲液洗珠。每个试管中加入1毫升缓冲液，旋转5-8分钟，在室温下，以800rpm旋转1分钟，然后弃上清。重复与高盐免疫复合物洗涤缓冲液和氯化锂的免疫复合物洗涤缓冲液洗一次，两次，共洗涤5次（ 表2）的TE缓冲液。 </li></ol> 7。洗脱<ol><li>由前一日的解冻输入样本与洗脱剂进行处理。 </li><li>洗脱缓冲新鲜（ 表3）。 </li><li> IP地址，并输入控制样品加1-2额外的样品需要足够的洗脱缓冲的预混液。 </li><li>加入100μl洗脱缓冲每个IP样本为15米，并在室温下孵育在旋转。 </li><li> ，持续1分钟，在800 rpm的转速下停止旋转，并添加新的管中的上清液。 </li><li>添加另一个珠每管100微升洗脱缓冲液，并在室温孵育15分钟旋转。 </li><li>涡旋混合15秒温育后，在5,000 rpm下旋转1分钟，然后结合上清液与从第一洗脱的上清液。 （请确定没有遗留上清中珠，如果不确定，降速再次上清液1分钟5000转，在一个新的管收集上清。 </li><li>洗脱缓冲液中加入180μl20μl的输入控制样品。 </li></ol> 8。反向交叉链接<ol><li> 200微升洗脱液和输入控件，添加8微升5 M氯化钠。 </li><li>在水浴中于65℃培养过夜，用封口膜和孵化的密封管。 </li></ol> 9。 DNA纯化<ol><li>对待每个样品1小时1微升的核糖核酸酶A在37°C。 </li><li> 4μ升的0.5M EDTA，8微升1M的Tris-HCl，混合，然后加入1微升的蛋白酶K在45℃下向每个样品，并培育2小时。 </li><li>用QIAquick PCR纯化试剂盒纯化的样品。样品可以保存在-20℃和PCR检查可以做到在稍后的日期。 </li></ol> *或者，芯片同类的磁珠可以用来代替琼脂糖免疫沉淀部分。 10。 PCR检查<ol><li>用于PCR检查，使用已知的约束感兴趣的蛋白质的不同地区的引物。此外，使用非结合区的引物作为阴性对照。 </li><li>对于该反应的试剂混合。按1:100稀释的输入样本（ 表3）。 </li><li>运行反应。 PCR程序： </li></ol> 第1步：94℃3分钟 第2步：94℃20秒 59℃30秒 72℃30秒 （CYCL至少30重复第2步ES） 第3步：72℃2分钟<ol start="4"><li> 1％琼脂糖凝胶上运行样本。 </li><li>也可确定定量实时PCR富集。 </li></ol>

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作者宣称，他们有没有竞争经济利益。

开展全基因组的蛋白-DNA相互作用协会使用的ChIP-seq的档案现在是可行的，因为已经非常最近展示了与其他转录因子2,3。一个成功的测序结果的关键是一个高品质的染色质免疫沉淀DNA模板的生成。 一旦DNA模板已生成并确定要充分丰富，可以把它在后续测序文库制备。例如，可以使用由供应商提供，Illumina公司的测序文库协议。这个库的大小选择，可以通过凝胶电泳和随?...

对于许多年一直存在的未满足的需要，以确定约束和受给定的蛋白质的基因组范围，特别是那些在转录因子类基因组。 奥多姆等[1]用染色质免疫沉淀技术（ChIP）结合系统识别占用由预先指定的转录调控因子，在人体的肝脏和胰岛基因的启动子微阵列。随后，约翰逊等人开发了大规模染色质免疫沉淀的基础上直接超高通量DNA测序技术（ChIP-seq的）为了全面映射整个哺乳动物基因组的蛋白-DNA相互作用。测试的情况下，他们在体内的结合映射神经元限制性沉默因子（NRSF）到1946年人类基因组中的位置。数据显示清晰分辨率装订位置（50个碱基对），这有利于双方的i染料溶液的图案和NRSF结合基序识别。这些芯片-Seq数据也有较高的敏感性和特异性和统计的信心（P &lt;10-4），推断新的候选相互作用是重要的属性。 Robertson 等人。3也可用于芯片起，以映射STAT1目标干扰素-γ（IFN-γ）刺激和未刺激的人HeLa S3细胞在体内 。芯片起，使用15.1和1290万唯一对应的序列读取和估计错误​​发现率小于0.001，他们确定了41,582和11,004公认的STAT1结合区在刺激和未刺激细胞，分别。已知含有STAT1干扰素反应的结合位点4-8的34个位点，芯片起发现有24人（71％）。芯片的seq目标富集序列与已知的STAT1结合基序。比较两个现有的芯片-PCR数据集建议该芯片起的灵敏度为70％和92％，特异度为至少95％之间。此外，它的ChIP-seq的很清楚，同时提供低复杂性和敏感性分析，随着测序深度。 由于这样，“下一代”的基因组测序技术提供了1-2个数量级的增加量的序列，可以产生成本效益比老技术9。因此，芯片的seq方法直接提供的全基因组覆盖率哺乳动物蛋白-DNA相互作用进行有效的分析。 在2006年，强烈关联，在7-2（TCF7L2）与2型糖尿病的基因转录因子的变种被发现10。其他研究者已经独立复制这一发现在不同的种族，有趣的是，从2型糖尿病全基因组关联研究发表在自然 11,12 </sTCF7L2高达13-15， 科学和其他地方的16,17，的确是最强的协会，这是目前被认为是最重要的2型糖尿病的遗传发现迄今18-20。此外，TCF7L2一直与癌症风险21,22，事实上，这方面变得更加明显8q24染色体位点，揭示了全基因组关联研究，许多癌症，包括大肠癌，被证明是由于极端的上游TCF7L2结合元件驱动MYC基因的转录23,24。因此，有极大的兴趣，确定下游受这个关键转录因子的基因。 基于与TCF7L2作为该方法的一个例子的经验，本文概述了如何产生高品质的芯片的DNA模板。大肠癌细胞株HCT116芯片，为了建立一个高甲阶酚醛树脂为后续测序ution地图基因TCF7L2 25约束的努力产生进一步了解其复杂性状的发病机制中的关键作用。

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td></td>
 </tr>
 <tr>
 <td>QIAquick PCR Purification Kit</td>
 <td>Qiagen</td>
 <td>28104 </td>
 <td></td>
 </tr>
 <tr>
 <td>EZ-ChIP Kit </td>
 <td>Millipore </td>
 <td>17-371 </td>
 <td></td>
 </tr>
 <tr>
 <td>GoTaq Hot Start Polymerase </td>
 <td>Promega</td>
 <td>M5001</td>
 <td></td>
 </tr>
 <tr>
 <td>Misonix Sonicator</td>
 <td>Qsonica </td>
 <td>XL-2000 </td>
 <td></td>
 </tr>
 <tr>
 <td>
 NanoDrop 1000 Spectrophotometer </td>
 <td>
 Thermo-Scientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Positive control primer sequences (TCF7L2-1) 
 Forward- 5'-TCGCCCTGTCAATAATCTCC-3' 
 Reverse- 5'-GCTCACCTCCTGTATCTTCG-3' 
 Negative control primer sequences (CTRL-1) 
 Forward-5'-ATGTGGTGTGGCTGTGATGGGAAC-3'
 
 Reverse- 5'-CGAGCAATCGGTAAATAGGTCTGG-3'</td>
 </tr>
 </tbody>
</table>

generation of high quality chromatin immunoprecipitation dna template for high-throughput sequencing (chip-seq)

芯片测序技术（ChIP-seq的）方法直接提供全基因组的覆盖面，结合染色质免疫沉淀技术（ChIP）和大规模并行测序可以用来识别剧目哺乳动物的DNA序列转录因子在体内的约束。 “下一代”的基因组测序技术提供了1-2个数量级的增加量序列，可以是成本有效的ChIP-seq的方法直接进行有效的分析哺乳动物全基因组覆盖率较老的技术，从而使生成的蛋白质-DNA相互作用。 对于成功的ChIP-seq的方法，必须产生高品质芯片的DNA为模板，以获得最佳的测序成果。的说明是基于经验，最强烈暗示发病机制中的2型糖尿病，即转录因子的转录因子7类2（TCF7L2基因的蛋白产物）。在各种癌症中，这一因素也被牵连。 概述是如何产生的，以高品质的芯片DNA模板来自大肠癌细胞株HCT116通过测序，以确定约束TCF7L2基因，进一步了解其发病机制中的关键作用，建立一个高解析度地图复杂性状。

已被超声处理的染色质，并用RNA酶和蛋白酶处理后，将样品在2％琼脂糖凝胶上运行的应出示涂抹标本所需的大小的DNA与散装。如果几个不同的周期进行测试，逐渐减少的周期数增加（ 图2）的大小应被看作是。 完成后的协议的免疫沉淀部的富集，可以由PCR或实时PCR检查。 PCR样品在琼脂糖​​凝胶上运行，应该有输入和芯片乐队（使用抗体蛋白的兴趣，这是在这?...

Watch this Scientific Journal Video about Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq at JoVE.com

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

染色质免疫沉淀和超高通量测序（芯片起）的组合可以识别并映射在一个给定的组织或细胞系的蛋白质-DNA相互作用。概述是如何产生一个高质量的ChIP随后的测序模板，使用的经验，例如转录因子TCF7L2。

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 ...

generation-high-quality-chromatin-immunoprecipitation-dna-template

Research

JoVE Journal

Biology

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

24.3K 次观看.  Children's Hospital of Philadelphia Research Institute. 染色质免疫沉淀和超高通量测序（芯片起）的组合可以识别并映射在一个给定的组织或细胞系的蛋白质-DNA相互作用。概述是如何产生一个高质量的ChIP随后的测序模板，使用的经验，例如转录因子TCF7L2。

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

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

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

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 ...

科研

生物学

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

有针对性的DNA甲基化分析通过新一代测序

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

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 ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

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

DamID-SEQ：蛋白质-DNA相互作用的全基因组映射腺嘌呤甲基化的DNA片段通过高通量测序

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

逆转录病毒整合位点的扩增，新一代测序和基因组DNA制图

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

高通量CRISPR载体的构建及DNA修饰的表征番茄毛状根的一代

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 &#946;-lactamase selected at a clinically relevant dosage of ampicillin are provided.

经过一蛋白饱和诱变图书馆的功能评价规范利用高通量测序

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

平行高通量单分子动力学测定，用于现场特异性DNA裂解

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...

Modified Methods for Loading of High-Throughput DNA Extraction Plates Reduce Potential for Contamination

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, host characteristics, and broader ecosystem functions. With this rapid increase in breadth and depth of sequencing capabilities have come methods to extract, amplify, analyze, and interpret environmental DNA successfully with maximum efficiency. Unfortunately, performing DNA extractions quickly can come at the cost of increasing the risk of contamination among samples. In particular, high-throughput extractions that are based on samples contained in a 96-well plate offer a relatively quick method, compared to single-tube extractions, but also increase opportunities for well-to-well cross-contamination. To minimize the risk of cross-contamination among samples, while retaining the benefits of high-throughput extraction techniques, we developed a new method for loading environmental samples into 96-well plates. We used pierceable PCR sealing films to cover each plate while loading samples and added samples first to PCR tubes before moving them into wells; together, these practices reduce the risk of sample drift and unintended double loading of wells. The method outlined in this paper provides researchers with an approach to maximize available high-throughput extraction techniques while reducing the risk of cross-contamination inherent to 96-well plates. We provide a detailed step by step outline of how to move from sample collection to DNA extraction while minimizing the risk of unwanted cross-contamination.

Current methods for loading 96-well plates for DNA extractions can be prone to contamination. We detail a new method for loading 96-well plates that reduces risk of cross-contamination among wells. Our method will help other researchers capitalize on the efficiency of high-throughput DNA extraction techniques and minimize risk of contamination.

改进的高通量DNA提取板加载方法，降低污染的可能性

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, ...