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TritonTM X-100

Triton TM X-100

Company: Sigma-Aldrich
Catalog#: T9284
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Adapting the Smart-seq2 Protocol for Robust Single Worm RNA-seq
Author:
Date:
2018-02-20
[Abstract]  Most nematodes are small worms that lack enough RNA for regular RNA-seq protocols without pooling hundred to thousand of individuals. We have adapted the Smart-seq2 protocol in order to sequence the transcriptome of an individual worm. While developed for individual Steinernema carpocapsae and Caenorhabditis elegans larvae as well as embryos, the protocol should be adaptable for other nematode species and small invertebrates. In addition, we describe how to analyze the RNA-seq results using the Galaxy online environment. We expect that this method will be useful for the studying gene expression variances of individual nematodes in wild type and mutant backgrounds. [摘要]  大多数线虫是小蠕虫,缺乏足够的RNA用于常规的RNA-seq协议,而没有汇集成千上万的个体。 我们已经调整了Smart-seq2协议来排序单个蠕虫的转录组。 虽然针对Steinernema carpocapsae和Caenorhabditis elegans幼虫以及胚胎开发,但该方案应该适用于其他线虫物种和小无脊椎动物。 另外,我们介绍如何使用Galaxy在线环境分析RNA-seq结果。 我们预计这种方法将有助于研究野生型和突变体背景个体线虫的基因表达差异。

【背景】低输入RNA-seq方案和扩增试剂盒,例如Smart-seq(Takara Bio,USA,Inc)和SuperAmp(Miltenyl Biotec,Inc),已经越来越多地开发和商业化,作为对低输入RNA-基于小组织,单一微生物和单细胞的seq研究。这些研究经常探索并解决特定群体(例如细胞群体,复杂组织或微生物群体)的个体中的异源基因表达。针对微生物(如线虫)的低输入RNA-seq方案的改进和适应将通过允许在单一线虫水平上分析基因表达异质性而极大地有益于线虫领域。在这里,我们已经调整了单细胞RNA-seq方案Smart-seq2(Picelli等人,2013和2014; Trombetta等人,2014),对于单线虫RNA测序。我们成功地在昆虫寄生线虫Steinernema ...

Mouse Satellite Cell Isolation and Transplantation
Author:
Date:
2018-01-20
[Abstract]  Satellite cell (SC) transplantation represents a powerful strategy to investigate SC biology during muscle regeneration. We described here a protocol for SC isolation from green fluorescent protein (GFP)-expressing mice and their transplantation into murine muscles. This procedure was originally used to assess the effects of the hormone unacylated ghrelin on muscle regeneration, in particular evaluating how the increase of unacylated ghrelin in the recipient muscle affected the engraftment of donor SCs (Reano et al., 2017). [摘要]  卫星细胞(SC)移植代表了肌肉再生期间SC生物学研究的强大策略。 我们在这里描述了从绿色荧光蛋白(GFP)表达的小鼠和他们的小鼠肌肉移植SC分离的协议。 该程序最初用于评估激素非酰化生长素释放肽对肌肉再生的影响,特别是评估受体肌肉中未酰化的生长素释放肽的增加如何影响供体SC的植入(Reano等人,2017年)。

【背景】由分化的肌纤维组成的骨骼肌可以在受伤时再生。肌肉再生依赖于居住在肌肉纤维基底层之下的称为卫星细胞(SC)的静止驻留干细胞群(Mauro,1961)。受伤后,SC经历激活,广泛的增殖,分化和融合,最终修复或替换受损的肌纤维(Collins等人,2005)。

由于移植的成肌细胞可以与宿主成肌细胞融合,因此SC的移植被认为是杜氏肌营养不良症(DMD)的潜在疗法多年的时间,这提示了在缺陷纤维中功能性修复的可能性(Partridge ...

High Throughput NPY-Venus and Serotonin Secretion Assays for Regulated Exocytosis in Neuroendocrine Cells
Author:
Date:
2018-01-05
[Abstract]  Here we describe two assays to measure dense core vesicle (DCV) exocytosis-mediated cargo secretion in neuroendocrine cells. To conduct siRNA screens for novel genes in regulated DCV exocytosis, we developed a plate reader-based secretion assay using DCV cargo, NPY-Venus, and an orthogonal 3H-serotonin secretion assay. The NPY-Venus secretion assay was successfully used for a high throughput siRNA screen, and the serotonin secretion assay was used to validate hits identified from the screen (Sorensen, 2017; Zhang et al., 2017). [摘要]  在这里我们描述了两种测定方法来测量神经内分泌细胞中的致密核心囊泡(DCV)胞吐作用介导的货物分泌。 为了在受调控的DCV胞吐作用中对新基因进行siRNA筛选,我们开发了使用DCV货物,NPY-金星以及正交3H-血清素分泌测定的基于读板器的分泌测定法。 NPY-金星分泌测定成功地用于高通量siRNA筛选,并且使用血清素分泌测定来验证从筛选中鉴定的命中(Sorensen,2017; Zhang等人,2017)。


【背景】致密核心囊泡(DCV)胞吐作用介导内分泌和神经内分泌细胞分泌蛋白质,肽和小分子。当蛋白质和肽激素被内质网上的核糖体合成时,蛋白质和肽激素进入分泌途径,并在跨高尔基网络(TGN)(Borgonovo等人,2006; Bowman等, >等人,2009)。小分子物质,如5-羟色胺,直接被胞质单胺转运蛋白(VMATs)从细胞质直接吸收到DCV的内腔(Sudhof,2004)。在非刺激条件下,货物分子被储存在靠近质膜的DCV内。当细胞接受增加细胞质Ca ...

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