| Low-cost and High-throughput RNA-seq Library Preparation for Illumina Sequencing from Plant Tissue
|
|
Author:
Date:
2020-10-20
[Abstract] Transcriptome analysis can provide clues to biological processes affected in different genetic backgrounds or/and under various conditions. The price of RNA sequencing (RNA-seq) has decreased enough so that medium- to large-scale transcriptome analyses in a range of conditions are feasible. However, the price and variety of options for library preparation of RNA-seq can still be daunting to those who would like to use RNA-seq for their first time or for a single experiment. Among the criteria for selecting a library preparation protocol are the method of RNA isolation, nucleotide fragmentation to obtain desired size range, and library indexing to pool sequencing samples for multiplexing. Here, we present a high-quality and a high-throughput option for preparing libraries from ...
[摘要] [摘要] 转录组分析可以为不同遗传背景或不同条件下的生物学过程提供线索。RNA测序（RNA-seq）的价格已经下降到足够低的程度，因此在各种条件下进行中大规模转录组分析是可行的。然而，对于那些希望第一次使用RNA-seq或进行单个实验的人来说，RNA-seq库制备的价格和各种选择仍然是令人望而生畏的。选择文库制备方案的标准包括RNA分离方法、核苷酸片段化以获得所需的大小范围，以及文库索引以汇集测序样本进行多路复用。在这里，我们提出了一个高质量和高通量的选择，从多聚腺苷酸mRNA制备文库用于转录组分析。高质量和高通量的方案选择都包括通过磁珠使poly-A尾部沉淀，cDNA合成，然后通过Tn5介导的“标记”同时裂解和添加适配器的步骤。该方案的所有步骤均已通过拟南芥叶片和幼苗组织的验证，并简化为协同工作，在资金和时间上成本最低，因此旨在为转录组分析提供一个初学者友好的从开始到完成的RNA序列库制备。
[背景] 通过Southern印迹、expressed sequence ...
|
|
|
| Isolation of Chromatin-bound Proteins from Subcellular Fractions for Biochemical Analysis
|
|
Author:
Date:
2018-10-05
[Abstract] Shuttling of proteins between different cellular compartments controls their proteostasis and can contribute in some cases to regulate their activity. Biochemical analysis of chromatin-bound proteins, such as transcription factors, is often difficult because of their low yield and due to the interference from nucleic acids. This protocol describes a method to efficiently fractionate cells combined with a mechanical (i.e., sonication) or an enzymatic treatment (i.e., benzonase) that facilitates analysis of chromatin-bound protein extracts by Western blot analysis or by protein pull-down assays. This approach can be valuable to enrich a particular protein within a particular subcellular fraction either to study specific post-translational modification patterns or to ...
[摘要] 在不同细胞区室之间穿梭蛋白质控制它们的蛋白质稳态，并且在某些情况下可以有助于调节它们的活性。 染色质结合蛋白（例如转录因子）的生化分析通常是困难的，因为它们的产率低并且由于核酸的干扰。 该协议描述了一种有效分离细胞的方法，结合机械（即，超声处理）或酶处理（即，benzonase），有助于分析染色质结合蛋白提取物 通过蛋白质印迹分析或蛋白质下拉分析。 该方法对于富集特定亚细胞级分内的特定蛋白质以研究特定的翻译后修饰模式或鉴定特定的蛋白质 - 蛋白质相互作用可能是有价值的。
【背景】许多染色质结合蛋白的活性和翻译后调节研究很少，因为在分离它们进行生化分析时存在技术困难。这甚至是转录因子的情况，例如基本的螺旋 - 环 - 螺旋（bHLH）转录因子，其通常在组织或细胞模型中具有稀缺的时间和空间表达模式（Dennis 等。，2018）。当生物材料的量成为研究分子途径的障碍时，协议细化有助于解除技术限制（Gillotin和Guillemot，2016）。在我们最近的研究中，我们努力了解神经元bHLH转录因子Ascl1的蛋白水解是如何在神经元分化的细胞模型中调节的（Gillotin et ...
|
|
|
| MicroScale Thermophoresis as a Tool to Study Protein-peptide Interactions in the Context of Large Eukaryotic Protein Complexes
|
|
Author:
Date:
2017-12-05
[Abstract] Protein-peptide interactions are part of many physiological processes, for example, epigenetics where peptide regions of histone complexes are crucial for regulation of chromatin structure. Short peptides are often also used as alternatives to small molecule drugs to target protein complexes. Studying the interactions between proteins and peptides is thus an important task in systems biology, cell biology, biochemistry, and drug design. However, this task is often hampered by the drawbacks of classical biophysical methods for analysis of molecular interactions like surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC), which require immobilization of the interaction partners or very high sample concentrations. MicroScale Thermophoresis (MST) is an innovative method ...
[摘要] 蛋白质 - 肽相互作用是许多生理过程的一部分，例如表观遗传学，其中组蛋白复合物的肽区域对于染色质结构的调节是至关重要的。短肽通常也被用作小分子药物靶向蛋白质复合物的替代物。研究蛋白质和肽之间的相互作用是系统生物学，细胞生物学，生物化学和药物设计中的重要任务。然而，这一任务往往受到经典生物物理学方法分析分子间相互作用的缺陷的困扰，例如表面等离子体共振（SPR）或等温滴定量热法（ITC），其需要固定相互作用配偶体或非常高的样品浓度。 MicroScale热泳（MST）是一种创新的方法，可以确定分子间相互作用的重要参数，如解离常数，化学计量和热力学。而且，它可以快速准确地进行，可以自由选择缓冲液或生物液体，不需要固定样品，样品消耗也非常少。这里我们详细描述了两个MST测定法，其分析（i）真核RNA聚合酶II的某些肽段与真核转录延伸复合物的蛋白质亚基之间的相互作用和（ii）N-末端组蛋白尾肽与表观遗传学之间的相互作用读者蛋白质。这些实验表明，MST能够表征蛋白质 - 肽相互作用，这些相互作用仅由肽的微小变化触发，例如，在特定的丝氨酸残基处仅有一个磷酸化。
【背景】生物学背景：蛋白质 - ...
|
|
|