| A Versatile Protocol to Quantify BCR-mediated Phosphorylation in Human and Murine B Cell Subpopulations
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Author:
Date:
2021-02-05
[Abstract] Signal transduction is the process by which molecular signals are transmitted from the cell surface to its interior, resulting in functional changes inside the cell. B cell receptor (BCR) signaling is of crucial importance for B cells, as it regulates their differentiation, selection, survival, cellular activation and proliferation. Upon BCR engagement by antigen several protein kinases, lipases and linker molecules become phosphorylated. Phosphoflow cytometry (phosphoflow) is a flow cytometry-based method allowing for analysis of protein phosphorylation in single cells. Due to recent advances in methodology and antibody availability – together with the relatively easy quantification of phosphorylation – phosphoflow is increasingly and more commonly used, compared to classical ...
[摘要] [背景] 信号转导是分子信号从细胞表面传递到细胞内部的过程，导致细胞内部功能发生变化。B细胞受体（BCR）信号对于B细胞至关重要，因为它调节了它们的分化，选择，存活，细胞活化和增殖。抗原与BCR结合后，一些蛋白激酶，脂肪酶和接头分子会被磷酸化。磷酸流式细胞仪（phosphoflow）是一种基于流式细胞仪的方法，可以分析单个细胞中的蛋白质磷酸化。由于方法学和抗体的可获得性的最新进展，再加上相对容易的磷酸化定量，与传统的蛋白质印迹分析相比，越来越多地使用磷酸流。但是，建立一种适用于所有感兴趣目标的方法可能具有挑战性。在这里，我们提出了逐步的磷流方案，允许对信号分子的磷酸化状态进行评估，并进行广泛的染色，以鉴定各种人和鼠B细胞亚群，如先前在Rip等人的原始论文。，（2020 ）。在原始论文中对磷流靶标的描述旁边，我们提供了在BCR信号传导中起关键作用的其他靶标的指导。循序渐进的磷酸流操作流程是用户友好的操作，可对人和鼠B细胞亚群中各种BCR信号分子的磷酸化进行灵敏检测。
[背景] ...
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| Low-cost and High-throughput RNA-seq Library Preparation for Illumina Sequencing from Plant Tissue
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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 ...
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