| Post-crystallization Improvement of RNA Crystals by Synergistic Ion Exchange and Dehydration
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Author:
Date:
2015-09-05
[Abstract] Compared to the recent dramatic growth in the numbers of genome-wide and functional studies of complex non-coding RNAs, mechanistic and structural analyses have lagged behind. A major technical bottleneck in the structural determination of large RNAs and their complexes is preparation of diffracting crystals. Empirically, a vast majority of such RNA crystals fail to diffract X-rays to usable resolution (~4 Å) due to their inherent disorder and non-specific packing within the crystals. Here, we present a protocol that combines post-crystallization cation replacement and dehydration that dramatically improved the diffraction quality of crystals of a large gene-regulatory mRNA-tRNA complex. This procedure not only extended the resolution limit of X-ray data from 8.5 to 3.2 Å, but also ...
[摘要] 与最近在复杂的非编码RNA的基因组范围和功能研究的数量的显着增长相比，机械和结构分析已经落后。在大RNA及其复合物的结构确定中的主要技术瓶颈是衍射晶体的制备。经验上，由于其固有的无序和在晶体内的非特异性堆积，绝大多数这样的RNA晶体不能将X射线衍射到可用的分辨率（〜4）。在这里，我们提出一个协议，结合后结晶阳离子替代和脱水，大大改善晶体的大基因调节mRNA-tRNA复杂的衍射质量。该程序不仅将X射线数据的分辨率极限从8.5扩展到3.2，而且还显着提高了数据的质量，实现了重新定相和结构确定。因为它利用了反离子和溶剂化在RNA结构中的一般重要性，这个程序可能证明在其他大型非编码RNA的晶体学分析中广泛有用。
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| Library Construction for Genome-wide Bisulfite Sequencing in Plants
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Author:
Date:
2013-12-20
[Abstract] DNA methylation is the most studied epigenetic modification, which involves the addition of a methyl group to the carbon-5 position of cytosine residues in DNA. DNA methylation is important for the regulation of gene expression. Bisulfite sequencing is the gold standard technique for determining genome-wide DNA methylation profiles in eukaryotes. This protocol describes how to prepare libraries of genomic DNA for whole-genome bisulfite sequencing in Arabidopsis, which could be adapted for use in other plant species.
[摘要] DNA甲基化是研究最多的表观遗传修饰，其涉及将甲基添加到DNA中胞嘧啶残基的碳-5位置。 DNA甲基化对于基因表达的调节是重要的。 亚硫酸氢盐测序是确定真核生物全基因组DNA甲基化谱的金标准技术。 该协议描述了如何在拟南芥中制备用于全基因组亚硫酸氢盐测序的基因组DNA文库，其可以适用于其他植物物种。
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