斯塔尔基金会奖（MQZ），美国国立卫生研究院授予ES017166（MQZ），美国国家科学基金会：授予DMS0906593（HX）。

1。准备输入文件BCP分析<ol><li>对齐短期运行（芯片和输入库）使用首选短读校准软件到相应的参考基因组测序读。映射的位置应该被转换为6列浏览器的可扩展的数据格式（BED）13（UCSC基因组浏览器， <a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu/</a> ），制表符分隔的每行对应的读表示映射的染色体，起始位置（从0开始），结束位置（半开），读的名字，得分（可选）和链。 </li></ol> 2A。弥漫读取配置文件：预处理芯片的读为检测弥漫数据丰富的群岛密度<ol><li>扩展到预定的片段长度， 即芯片和输入对应的位置。针对在酶消化或超声处理的DNA的片段大小，通常为约200 bp的。片段数aggrega特德在相邻位置。 bin大小设置为默认情况下，估计片段长度为200 bp的。 </li><li>任何可能的变化点中的一组的箱具有相同的读取计数将最有可能下降在最的外边界。因此，这是不可能的，将发生在两个时间窗相同的读计数的内部边界之间的变化点。因此，组相邻位置，以相同的读取每个回收箱，成一个单一的块， 即 。 bedGraph格式13。 </li></ol> 2B。点状读取配置文件：预处理芯片和输入BED检测的峰点状数据文件<ol><li>总重叠读取，读取分别为正，负链芯片。链特定的读的密度应形成一个加号和减号峰双峰档案。选择加/减对最丰富的山峰和使用首脑会议库片段长度的估计值之间的距离。 </li><li>按住Shift键的芯片和输入读取的片段文件ngth的中心，并重新计算的转移和合并的加号和减号链读取读取密度。这种推定 ​​片段长度的方法是采用从章， 等。3。应分组具有相同的合并计数的位置，成块，类似于步骤2A.2。 </li></ol> 3。估算后，平均密度每块使用我们的BCMIX逼近<ol><li>读取每个块的密度建模为泊松分布，泊松（θt）的，与平均参数Gamma分布的混合物后，Γ（α，β），和先验概率的变化点发生在任何数据块边界空调泊松（θ 吨 ）G（α，β）。有效的渲染无限的状态HMM模型。估价超参数，α，β， 和 p，使用最大后验似然。 </li><li>显式计算的贝叶斯估计每个块，θ 吨 ， 为E（θ 吨|γZ）。更换更传统，但耗费时间向前和向后的过滤器经常使用的HMM模型，更有效地计算界的复杂混合近似估计后验手段，θC。后的手段，将“平滑”成一个近似分段恒定的个人资料，以便块相同，θC，应进一步封锁一起更新的边界坐标。 </li></ol> 4A。成段弥漫性增高，弥漫读取配置文件：后处理后的手段<ol><li>使用数字输入读取每一个新的θC座为背景率，泊松（λ 一 ）每使用一个简单的假设检验的基础上的芯片是否后平均，θC，超过某个阈值δ确定富集。 90 日&lt;/ SUP&gt;位数是的默认d和在大多数情况下是合适的。 </li><li>合并相邻的θC块，超过了浓缩成一个单一的地区和报表合并简单的床格式的坐标。或者，用户可以为每个块在bedGraph格式保持高分辨率的细节的读出的密度估计θc的报告。 </li></ol> 4B。点状到山顶考生的阅读概况：后处理后的手段<ol><li>定义背景率，泊松（λ 一 ），作为平均的所有读出的计数（γ2）和拣选超过阈值，D的所有块。由于点状峰预期更基本上富集，默认δ被设置为99 次泊松（λ 一 ） -分位数。 </li><li>设置块最大的θc为候选峰值首脑会议的毗邻的侧翼块共享一个类似的读巢穴SITY（±1读取数允许有轻微的变化）。此邻接的候选结合位点的区域被定义为。 </li><li>计算λ2作为沉淀的候选结合位点与假设检验的平均读取计数，这与输入的背景是零假设H 0，λ1≥λ2和拒绝 H 0的基础上的p-值的阈值。输出候选人峰在床上格式。 </li></ol>

<ol>
	<li>Park, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIP-seq:+advantages+and+challenges+of+a+maturing+technology.">ChIP-seq: advantages and challenges of a maturing technology.</a> Nat. Rev. Genet. 10, 669-680 (2009).</li><li>Barski, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+profiling+of+histone+methylations+in+the+human+genome.">High-resolution profiling of histone methylations in the human genome.</a> Cell. 129, 823-837 (2007).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model-based+Analysis+of+ChIP-Seq+(MACS).">Model-based Analysis of ChIP-Seq (MACS).</a> Genome Biol. 9, R137 (2008).</li><li>Zang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clustering+approach+for+identification+of+enriched+domains+from+histone+modification+ChIP-Seq+data.">A clustering approach for identification of enriched domains from histone modification ChIP-Seq data.</a> Bioinformatics. 25, 1952-1958 (2009).</li><li>Jothi, R., Cuddapah, S., Barski, A., Cui, K., Zhao, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+identification+of+in+vivo+protein-DNA+binding+sites+from+ChIP-Seq+data.">Genome-wide identification of in vivo protein-DNA binding sites from ChIP-Seq data.</a> Nucleic Acids Res. 36, 5221-5231 (2008).</li><li>Qin, Z. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HPeak:+an+HMM-based+algorithm+for+defining+read-enriched+regions+in+ChIP-Seq+data.">HPeak: an HMM-based algorithm for defining read-enriched regions in ChIP-Seq data.</a> BMC Bioinformatics. 11, 369 (2010).</li><li>Song, Q., Smith, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+dispersed+epigenomic+domains+from+ChIP-Seq+data.">Identifying dispersed epigenomic domains from ChIP-Seq data.</a> Bioinformatics. 27, 870-871 (2011).</li><li>Spyrou, C., Stark, R., Lynch, A. G., Tavar&#233;, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BayesPeak:+Bayesian+analysis+of+ChIP-seq+data.">BayesPeak: Bayesian analysis of ChIP-seq data.</a> BMC Bioinformatics. 10, 299 (2009).</li><li>Lai, T., Xing, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+Bayesian+approach+to+multiple+change-points.">A simple Bayesian approach to multiple change-points.</a> Statistica Sinica. , (2011).</li><li>Robertson, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+profiles+of+STAT1+DNA+association+using+chromatin+immunoprecipitation+and+massively+parallel+sequencing.">Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing.</a> Nat. Methods. 4, 651-657 (2007).</li><li>Stitzel, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+epigenomic+analysis+of+primary+human+pancreatic+islets+provides+insights+into+type+2+diabetes+susceptibility+loci.">Global epigenomic analysis of primary human pancreatic islets provides insights into type 2 diabetes susceptibility loci.</a> Cell Metab. 12, 443-455 (2010).</li><li>Bernstein, B. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NIH+Roadmap+Epigenomics+Mapping+Consortium.">The NIH Roadmap Epigenomics Mapping Consortium.</a> Nat. Biotechnol. 28, 1045-1048 (2010).</li><li>Karolchik, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+UCSC+Table+Browser+data+retrieval+tool.">The UCSC Table Browser data retrieval tool.</a> Nucleic Acids Res. 32, 493-496 (2004).</li><li>Matys, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRANSFAC:+transcriptional+regulation,+from+patterns+to+profiles.">TRANSFAC: transcriptional regulation, from patterns to profiles.</a> Nucleic Acids Res. 31, 374-378 (2003).</li><li>Portales-Casamar, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JASPAR+2010:+the+greatly+expanded+open-access+database+of+transcription+factor+binding+profiles.">JASPAR 2010: the greatly expanded open-access database of transcription factor binding profiles.</a> Nucleic Acids Res. 38, D105-D110 (2010).</li></ol>

没有利益冲突的声明。

我们的目标是建立一个模型分析ChIPseq数据同样可以识别点状和弥漫性两种数据结构。到现在为止，富集的地区，尤其是弥漫性的地区，这反映了先决条件预期的大岛，大小，已经难以辨认。为了解决这些问题，我们利用最新进展在HMM的技术，具有许多优点，现有的的的启发式模型和缺乏创新的HMM模型。 我们的模型使用明确的公式与贝叶斯框架。从其他HMM模型，这是一个关键?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>基于Linux的工作站</td><td></td><td></td><td></td></tr></tbody></table>

a novel bayesian change-point algorithm for genome-wide analysis of diverse chipseq data types

ChIPseq是一种广泛使用的技术，用于调查蛋白质-DNA相互作用。读密度分布所产生的使用下一个 - 蛋白结合的DNA测序和对准读取到参考基因组的短。富集的区域显示峰，这往往显着不同的形状，这取决于对目标蛋白1。例如，转录因子通常在现场和序列特异性方式结合，往往会产生点状的山峰，而组蛋白修饰更普遍的特点是广泛的，弥漫性的岛屿富集2。可靠地识别这些地区是我们的工作重点。 算法分析ChIPseq数据采用各种方法，启发式3-5进行更加严格的统计模型， 例如隐马尔可夫模型（HMM模型）6-8。我们试图最小化的必要性难以界定，专案参数，通常的解决方案，妥协的分辨率和减轻直观的工具的可用性。基于HMM的方法，我们的目的是限制参数估计的程序和简单的，有限状态分类，往往利用。 此外，传统ChIPseq的数据分析包括分类的读取密度分布为点状或弥漫性的后续应用适当的工具。我们还旨在取代这两个不同的模型需要一个单一的，更灵活的模型，它可以足够能力解决整个频谱的数据类型。 为了实现这些目标，我们首先构建了一个统计框架，自然为蓝本ChIPseq数据结构尖端提前HMM模型9，利用唯一明确的公式，其性能优势的关键创新。更复杂的启发式模型，通过我们的的HMM可容纳无限的隐藏状态贝叶斯模型。我们把它应用在读取密度，进一步定义丰富的段，确定合理的变化点。我们的分析表明，我们的贝叶斯变点（BCP）算法具有降低计算复杂度，证明了一个简化的运行时间和内存占用。 BCP算法已成功地应用于斑点状的峰值和漫岛的识别与强​​大的精度和有限的用户定义的参数。这所示，它的多功能性和易用性。因此，我们认为它可以容易地实现在广泛的范围内的数据类型和最终用户的方式，很容易比较和对比，使其成为一个伟大的工具ChIPseq数据的分析，可以帮助研究团体之间的协作和佐证。在这里，我们演示了应用程序的BCP现有的转录因子10,11和表观数据来说明它的用处。

BCP擅长识别组蛋白修饰数据的广泛富集的地区。作为一个参照点，我们以前相比，我们的研究结果的SICER 3，现有的工具，已显示出强大的性能。为了更好地说明BCP的优势，我们研究了组蛋白的修饰，得到了很好的研究，建立评估成功率的基础。然后考虑到这一点，我们分析H3K36me3，因为它已经显示出强烈的积极转录基因体（ 图1）关联。相反，H3K36me3也被证明是互斥H3K27me3压?...

Watch this Scientific Journal Video about A Novel Bayesian Change-point Algorithm for Genome-wide Analysis of Diverse ChIPseq Data Types at JoVE.com

A Novel Bayesian Change-point Algorithm for Genome-wide Analysis of Diverse ChIPseq Data Types

我们的贝叶斯变点（BCP）算法的基础上通过隐马尔可夫模型的造型变化点的国家的最先进的进步和应用染色质免疫沉淀测序（ChIPseq）数据分析。 BCP执行在广泛和点状数据类型，但擅长准确地识别健壮的，可重复的岛屿弥漫组蛋白富集。

一种新型的全基因组分析的贝叶斯变点算法的多元ChIPseq数据类型

ChIPseq is a widely used technique for investigating protein-DNA interactions. Read density profiles are generated by using next-sequencing of ...

a-novel-bayesian-change-point-algorithm-for-genome-wide-analysis

Research

JoVE Journal

Biology

11.2K 次观看.  Stony Brook University. 我们的贝叶斯变点（BCP）算法的基础上通过隐马尔可夫模型的造型变化点的国家的最先进的进步和应用染色质免疫沉淀测序（ChIPseq）数据分析。 BCP执行在广泛和点状数据类型，但擅长准确地识别健壮的，可重复的岛屿弥漫组蛋白富集。

视频: A Novel Bayesian Change-point Algorithm for Genome-wide Analysis of Diverse ChIPseq Data Types

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

科研

生物学

Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both prokaryotic and eukaryotic cells lead to translational regulation which is a major cellular mechanism of stress response. Familiar examples are the stringent response in E. coli and the Gcn2 stress response pathway in yeast ([2-6]). Recent work in E. coli and S. cerevisiae have shown that tRNA charging patterns are highly dynamic and depends on the type of stress experienced by cells [1, 6, 7]. The highly dynamic, variable nature of tRNA charging makes it essential to determine changes in tRNA charging levels at the genomic scale, in order to fully elucidate cellular response to environmental variations. In this review we present a method for simultaneously measuring the relative charging levels of all tRNAs in S. cerevisiae . While the protocol presented here is for yeast, this protocol has been successfully applied for determining relative charging levels in a wide variety of organisms including E. coli and human cell cultures[7, 8].

Genome-wide Analysis of Aminoacylation Charging Levels of tRNA Using Microarrays

We describe a method for microarray analysis to determine relative aminoacylation levels of all tRNAs from S. cerivisiae.

（充电）酰化tRNA的使用微阵列技术水平的全基因组分析

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both ...

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

采用芯片识别亚型特定的靶基因的全基因组分析

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details).
To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3&#900;TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.

miRNA的目标使用的任务目标ID库的全基因组屏幕

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

定量和自动化高通量的全基因组RNAi屏幕 C。线虫</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

全基因组基因缺失<em&gt;链球菌血统</em通过高通量PCR

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

多聚核糖体分离和哺乳动物Translatomes分析在基因组范围内

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

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).

药物与DNA相互作用的细胞全基因组映射与COSMIC（小分子交联，染色质隔离）

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

从真核细胞染色体外的环状DNA基因组范围内的净化

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...

Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the prediction of family functions based on several features, such as the presence of sequence motifs or of particular sites for post-translational modification and the expression profile of family members in different conditions. This work describes a detailed protocol for gene family characterization. Here, the procedure is applied to the characterization of the Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligase family in grapevine. The methods include the genome-wide identification of family members, the characterization of gene localization, structure, and duplication, the analysis of conserved protein motifs, the prediction of protein localization and phosphorylation sites as well as gene expression profiling across the family in different datasets. Such procedure, which could be extended to further analyses depending on experimental purposes, could be applied to any gene family in any plant species for which genomic data are available, and it provides valuable information to identify interesting candidates for functional studies, giving insights into the molecular mechanisms of plant adaptation to their environment.

This article describes the procedure for the identification and characterization of a gene family in grapevine applied to the family of Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligases.

葡萄全基因组鉴定与表达的综合工作流 E3 泛素连接基因家族的 Meta 分析

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the ...