| Tethered Chromosome Conformation Capture Sequencing in Triticeae: A Valuable Tool for Genome Assembly
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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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| RNA Immunoprecipitation (RIP) Sequencing of Pri-miRNAs Associated with the Dicing Complex in Arabidopsis
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Author:
Date:
2018-07-05
[Abstract] RNA immunoprecipitation (RIP) is an antibody-based technique used to map in vivo RNA-protein interactions. DBR1, an RNA debranching enzyme, is responsible for the debranching of lariat RNA, for the degradation and turnover of lariat RNAs. It is well known that primary miRNA (Pri-miRNA) is recognized and further processed into mature miRNA by the Dicing complex mainly composed of DCL1 and HYL1. Due to the low abundance of pri-miRNAs, RIP followed qRT-PCR has been widely used to evaluate the binding efficiency of the Dicing complex with pri-miRNAs in previous studies. Therefore, the genome-wide evaluation of the Dicing complex with pri-miRNAs is lacking. With the improvement of high-throughput sequencing technologies, we successfully used RIP-seq to compare the binding efficiency ...
[摘要] RNA免疫沉淀（RIP）是一种基于抗体的技术，用于绘制体内 RNA-蛋白质相互作用。 DBR1是一种RNA脱支酶，负责套索RNA的脱支，用于套索RNA的降解和转换。众所周知，主要miRNA（Pri-miRNA）被主要由DCL1和HYL1组成的切割复合物识别并进一步加工成成熟miRNA。由于pri-miRNA的丰度较低，RIP随后的qRT-PCR已被广泛用于评估切割复合物与pri-miRNA在先前研究中的结合效率。因此，缺乏对具有pri-miRNA的切割复合物的全基因组评估。随着高通量测序技术的改进，我们成功地使用RIP-seq比较了Dicing复合物与野生型和 dbr1-2 突变体之间的pri-miRNA的结合效率。 。在该方案中，我们提供了在两种不同基因型之间的HYL1-YFP和DCL1-YFP转基因植物中使用GFP捕获珠的RIP-seq的详细描述。该方法可用于评估pri-miRNA与拟南芥中的切割复合物的结合，并且它可以应用于植物中的其他RNA结合蛋白。
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