| Bimolecular Fluorescence Complementation (BiFC) for Studying Sarcomeric Protein Interactions in Drosophila
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Author:
Date:
2020-04-05
[Abstract] Protein-protein interactions in Drosophila myofibrils are essential for their function and formation. Bimolecular Fluorescence Complementation (BiFC) is an effective method for studying protein interactions and localization. BiFC relies on the reconstitution of a monomeric fluorescent protein from two half-fragments when in proximity. Two proteins tagged with the different half-fragments emit a fluorescent signal when they are in physical contact, thus revealing a protein interaction and its spatial distribution. Because myofibrils are large networks of interconnected proteins, BIFC is an ideal method to study protein-protein interactions in myofibrils. Here we present a protocol for generating transgenic flies compatible with BiFC and a method for analyzing protein-protein ...
[摘要] [摘要] 果蝇肌原纤维中的蛋白质-蛋白质相互作用对其功能和形成至关重要。双分子荧光互补（BiFC ）是研究蛋白质相互作用和定位的一种有效方法。BiFC 依赖于邻近时从两个半片段重构单体荧光蛋白。标记有不同半片段的两种蛋白质在物理接触时会发出荧光信号，从而揭示了蛋白质相互作用及其空间分布。因为肌原纤维相互连接的蛋白质的大型网络中，附设是一种理想的方法来 研究肌原纤维中蛋白质之间的相互作用。在这里，我们提出了一种生成与BiFC 兼容的转基因果蝇的协议，以及一种基于肌原纤维中荧光BiFC 信号的蛋白质-蛋白质相互作用分析方法。我们的方案适用于大多数果蝇蛋白，只需稍加修改即可用于研究任何组织。
[背景] 肉瘤是横纹肌中最小的收缩单位，并沿着肌原纤维的长度以重复的方式延伸（Reedy和Beall，1993）。肉瘤产生肌肉收缩的能力取决于两个肌原纤维成分：细丝和粗丝。肌球蛋白粗丝固定在肌节中心的M线，而肌动蛋白细丝固定在肌节两侧的Z盘上。因此，Z盘对于维持肌原纤维的结构和收缩至关重要，而Z盘无法形成可能导致各种人类肌病的严重缺陷性肌肉表型（Lemke和Schnorrer，2017年）。
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| Human, Bacterial and Fungal Amplicon Collection and Processing for Sequencing
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Author:
Date:
2015-05-20
[Abstract] Sequencing taxonomic marker genes is a powerful tool to interrogate the composition of microbial communities. For example, bacterial and fungal community composition can be evaluated in parallel using the 16S ribosomal RNA gene for bacteria or the internal transcribed spacer region in fungi. These are conserved regions that are universal to a taxonomic clade, yet have undergone some degree of evolution such that different lineages can be differentiated. Conserved regions are used for design of universal priming sites that allow amplification of the marker gene out of a mixed microbial community. Here, we describe our standard operating procedure to collect and sequence 16S rRNA and ITS1 amplicons from human skin. We use the 16S rRNA V1-V3 region for skin samples, as it has greater power ...
[摘要] 测序分类标记基因是一个强有力的工具，以询问微生物群落的组成。 例如，可以使用细菌的16S核糖体RNA基因或真菌中的内部转录间隔区平行评估细菌和真菌群落组成。 这些是对分类学分支是通用的保守区，但已经经历了某种程度的进化，使得不同谱系可以分化。 保守区用于设计允许从混合微生物群落扩增标记基因的通用引发位点。 在这里，我们描述我们的标准操作程序收集和序列16S rRNA和ITS1扩增子从人类皮肤。 我们使用16S rRNA V1-V3区域的皮肤样本，因为它有更大的权力分类皮肤中的常见葡萄球菌。 该方案适用于扩增子的454焦磷酸测序。
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| RNA-Seq Library Generation from Rare Human Cells Isolated by FACS
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Author:
Date:
2013-06-20
[Abstract] High throughput RNA Sequencing has revolutionized transcriptome analyses. However, most available protocols require micrograms of RNA rendering this technique not feasible for analyzing small numbers of cells, including precious rare cell types isolated from human tissues or organs. Here, we used an RNA Amplification System and describe a method for preparing RNA sense-strand cDNA libraries compatible with an Illumina sequencing platform starting from limited numbers of human fetal germ cells as well as human embryonic stem cells (hESCs) isolated using Fluorescence Activated Cell Sorting (FACS). With this protocol we generated seven RNA-Seq libraries starting from 4,000 germ cells sorted from fetal ovaries (n = 2) and fetal testes (n = 2) at 16-16.5 weeks of development and 4,000 sorted ...
[摘要] 高通量RNA测序革新了转录组分析。 然而，大多数可用的协议需要微克RNA，使得这种技术不可能用于分析少量的细胞，包括从人体组织或器官分离的珍贵的稀有细胞类型。 在这里，我们使用RNA扩增系统，并描述了一种制备RNA有义链cDNA文库的方法，其与Illumina测序平台相容，从有限数目的人胎儿生殖细胞以及使用荧光活化细胞分离的人胚胎干细胞（hESC） 排序（FACS）。 使用这个协议，我们生成七个RNA Seq库开始从4,000生殖细胞从胎儿卵巢（n = 2）和胎儿睾丸（n = 2）在16-16.5周的发展和4,000分选hESCs（n = 3）排序。 我们预测多重文库也可以通过用多重兼容的3'衔接子和索引的PCR引物替换这里使用的单重3'衔接子来产生。
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