| Preparation of Sequencing RNA Libraries through Chemical Cross-linking Coupled to Affinity Purification (cCLAP) in Saccharomyces cerevisiae
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Author:
Date:
2018-10-05
[Abstract] Ribonucleoprotein particles (mRNPs) are complexes consisting of mRNAs and RNA-binding proteins (RBPs) which control mRNA transcription localization, turnover, and translation. Some mRNAs within the mRNPs have been shown to undergo degradation or storage. Those transcripts can lack general mRNA elements, like the poly(A) tail or 5’ cap structure, which prevent their identification through the application of widely-used approaches like oligo(dT) purification. Here, we describe a modified cross-linking affinity purification protocol (cCLAP) based on existing cross-linking and immunoprecipitation ...
[摘要] 核糖核蛋白颗粒（mRNP）是由mRNA和RNA结合蛋白（RBP）组成的复合物，其控制mRNA转录定位，转换和翻译。已显示mRNP内的一些mRNA经历降解或储存。那些转录物可能缺乏一般的mRNA元件，如poly（A）尾或5'帽结构，这通过应用广泛使用的方法如oligo（dT）纯化来阻止它们的鉴定。在这里，我们描述了基于现有的交联和免疫沉淀（CLIP）方法的修饰的交联亲和纯化方案（cCLAP），以分离mRNP中可被去腺苷酸化，去除和/或部分降解的mRNA，从而开启了检测不同的可能性。非编码RNA（ncRNA）的类型。分离后，将RNA进行衔接子连接，然后进行下一代测序（NGS）。由于快速有效的交联和淬灭步骤，该方案也适用于瞬时诱导的mRNP颗粒。实例包括由外在应激物触发的处理体（PB）或应力颗粒（SG）。其重现性和广泛应用使该方案成为研究特定RNP的RNA组成的有用且有力的工具。
【背景】mRNP内转录物的表征对于理解细胞转录和转录后过程至关重要。通过交联和免疫沉淀，然后通过RNA-Seq从mRNP颗粒中分离RNA已经成为鉴定mRNA靶标的常用方法（Tagwerker et al。，2006; Hafner et al。，2010; Kishore et al。，2011）。 ...
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| Lectin Binding Analysis of Streptococcus mutans Glycoproteins
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Author:
Date:
2015-04-05
[Abstract] Bacterial glycoproteins are of increasing interest due to their abundance in nature and importance in health and infectious diseases. However, only a very small fraction of bacterial glycoproteins have been characterized and its post-translational modification machinery identified. While analysis of glycoproteins can be achieved through various techniques, this is often limited by the specific characteristics of individual proteins such as type and level of glycosylation. Lectins are sugar-binding proteins that recognize specific glycoconjugates in a manner similar to antigen-antibody ...
[摘要] 细菌糖蛋白由于其在自然界中的丰富性和在健康和传染病中的重要性而日益受到关注。 然而，只有非常小部分的细菌糖蛋白已被表征和其翻译后修饰机制鉴定。 尽管通过各种技术可以实现糖蛋白的分析，但是这常常受到单个蛋白质的特定特征的限制，例如糖基化的类型和水平。 凝集素是以类似于抗原 - 抗体相互作用的方式识别特异性糖缀合物的糖结合蛋白。 在这里，我们描述了一种简单的方法使用基于凝集素的蛋白质印迹分析检测糖蛋白，这可以应用于不同的生物体，并与各种其他策略的补充分析。
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| Preparation of Multiplexed Small RNA Libraries from Plants
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Author:
Date:
2014-11-05
[Abstract] High-throughput sequencing is a powerful tool for exploring small RNA populations in plants. The ever-increasing output from an Illumina Sequencing System allows for multiplexing multiple samples while still obtaining sufficient data for small RNA discovery and characterization. Here we describe a protocol for generating multiplexed small RNA libraries for sequencing up to 12 samples in one lane of an Illumina HiSeq System single-end, 50 base pair run. RNA ligases are used to add the 3’ and 5’ adaptors to purified small RNAs; ligation products that lack a small RNA molecule (adaptor-adaptor ...
[摘要] 高通量测序是探索植物中小RNA群体的强大工具。 来自Illumina测序系统的不断增加的输出允许多重多个样品，同时仍然获得足够的数据用于小RNA发现和表征。 在这里我们描述了一个协议，用于生成多重小RNA文库，用于在Illumina HiSeq系统单端，50碱基对运行的一个泳道中测序多达12个样品。 RNA连接酶用于将3'和5'接头添加至纯化的小RNA; 缺少小RNA分子（衔接子 - 衔接子产物）的连接产物被故意耗尽。 cDNA合成后，线性PCR步骤扩增DNA片段。 本文使用的3'PCR引物包括独特的6-核苷酸序列，以允许多达多达12个样品。
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