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Agencourt AMPure XP, 60 mL

Agencourt AMPure XP - PCR纯化

Company: Beckman Coulter
Catalog#: A63881
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Random Insertional Mutagenesis of a Serotype 2 Dengue Virus Clone
Author:
Date:
2018-08-20
[Abstract]  Protein tagging is a powerful method of investigating protein function. However, modifying positive-strand RNA virus proteins in the context of viral infection can be particularly difficult as their compact genomes and multifunctional proteins mean even small changes can inactivate or attenuate the virus. Although targeted approaches to functionally tag viral proteins have been successful, these approaches are time consuming and inefficient. A strategy that has been successfully applied to several RNA viruses is whole-genome transposon insertional mutagenesis. A library of viral genomes, each containing a single randomly placed small insertion, is selected by passaging in cell culture and the insertion sites can be identified using Next Generation Sequencing (NGS). Here we describe a ... [摘要]  蛋白质标记是研究蛋白质功能的有效方法。然而,在病毒感染的情况下修饰正链RNA病毒蛋白可能特别困难,因为它们的紧密基因组和多功能蛋白意味着即使很小的变化也可以使病毒失活或减弱。尽管功能性标记病毒蛋白的靶向方法已经成功,但这些方法耗时且效率低。已经成功应用于几种RNA病毒的策略是全基因组转座子插入诱变。通过细胞培养中的传代选择病毒基因组文库,每个文库含有单个随机放置的小插入,并且可以使用下一代测序(NGS)鉴定插入位点。在这里,我们描述了用于登革病毒16681株血清型2的转座子诱变的方案。含有短随机放置插入物的突变登革病毒文库通过哺乳动物细胞传代,插入由有活力后代的NGS定位。该方案分为四个阶段:登革热cDNA克隆的转座子诱变,病毒基因组转染到允许细胞,分离病毒后代基因组和测序文库制备。

【背景】 ...

Quantitative ChIP-seq by Adding Spike-in from Another Species
Author:
Date:
2018-08-20
[Abstract]  Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a routine procedure in the lab; however, epigenome-wide quantitative comparison among independent ChIP-seq experiments remains a challenge. Here, we contribute an experimental protocol combined with a computational workflow allowing quantitative and comparative assessment of epigenome using animal tissues. [摘要]  染色质免疫沉淀,然后测序(ChIP-seq)是实验室中的常规程序; 然而,独立ChIP-seq实验之间的表观基因组范围的定量比较仍然是一个挑战。 在这里,我们提供了一个实验方案,结合计算工作流程,允许使用动物组织定量和比较评估表观基因组。

【背景】 修饰组蛋白的染色质和表观遗传复合物调节DNA对转录机制的可及性,从而允许直接控制基因表达。为了表征组蛋白修饰的表观基因组特征,染色质免疫沉淀然后测序(ChIP-seq)已成为一种广泛使用的方法。然而,传统的ChIP-seq方案本质上不是定量的,因此禁止直接比较源自不同细胞类型的样品或经历过不同遗传或化学扰动的细胞。尽管已经提出了几种 in silico 归一化方法来克服这个缺点,但仍然缺乏基于实验的策略。 2014年,奥兰多等人(2014)开发了一种名为ChIP的方法,该方法使用参考外源基因组(ChIP-Rx),该方法利用恒定量的参考或''spike-in''表观基因组基于细胞的表观基因组比较。在当前的协议中,我们通过使用映射的尖峰参考表观基因组的百分比来改进该方法。我们已成功应用此协议直接比较来自动物组织的两个或更多ChIP-seq数据集。

Tethered Chromosome Conformation Capture Sequencing in Triticeae: A Valuable Tool for Genome Assembly
Author:
Date:
2018-08-05
[Abstract]  Chromosome conformation capture sequencing (Hi-C) is a powerful method to comprehensively interrogate the three-dimensional positioning of chromatin in the nucleus. The development of Hi-C can be traced back to successive increases in the resolution and throughput of chromosome conformation capture (3C) (Dekker et al., 2002). The basic workflow of 3C consists of (i) fixation of intact chromatin, usually by formaldehyde, (ii) cutting the fixed chromatin with a restriction enzyme, (iii) religation of sticky ends under diluted conditions to favor ligations between cross-linked fragments or those between random fragments and (iv) quantifying the number of ligations events between pairs of genomic loci (de Wit and de Laat, 2012). In the original 3C protocol, ligation frequency was ... [摘要]  染色体构象捕获测序(Hi-C)是一种全面询问细胞核中染色质三维定位的有效方法。 Hi-C的发展可以追溯到染色体构象捕获的分辨率和通量的连续增加(3C)(Dekker et al。,2002)。 3C的基本工作流程包括(i)通常用甲醛固定完整的染色质,(ii)用限制酶切割固定的染色质,(iii)在稀释条件下重新连接粘性末端,以促进交联片段之间的连接或随机片段之间的那些和(iv)量化基因组基因座对之间的连接事件的数量(de Wit和de Laat,2012)。在最初的3C方案中,通过半定量PCR扩增对应于少量基因组位点(“一对一”)的选定连接接头来测量连接频率(Dekker et al。,2002 )。然后,染色体构象捕获芯片(4C)和染色体构象捕获碳复制(5C)技术扩展3C以分别以“一对多”或“多对多”方式计算结扎事件。 Hi-C(Lieberman-Aiden et al。,2009)最终将3C与下一代测序相结合(Metzker,2010)。此处,在再连接之前,用生物素标记的核苷酸类似物填充粘性末端以在后续步骤中富集具有连接连接的片段。然后对Hi-C文库进行高通量测序,并将得到的读数映射到参考基因组,允许以“多对多”方式确定接触概率,其分辨率仅受限制性位点的分布限制和阅读深度。 Hi-C的首次应用是阐明人类基因组中的全球染色质折叠原理(Lieberman-Aiden et ...

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