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SuperScriptTM II Reverse Transcriptase

SuperScript ® II Reverse Transcriptase

Company: Thermo Fisher Scientific
Catalog#: 18064014
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A Small RNA Isolation and Sequencing Protocol and Its Application to Assay CRISPR RNA Biogenesis in Bacteria
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Date:
2018-02-20
[Abstract]  Next generation high-throughput sequencing has enabled sensitive and unambiguous analysis of RNA populations in cells. Here, we describe a method for isolation and strand-specific sequencing of small RNA pools from bacteria that can be multiplexed to accommodate multiple biological samples in a single experiment. Small RNAs are isolated by polyacrylamide gel electrophoresis and treated with T4 polynucleotide kinase. This allows for 3’ adapter ligation to CRISPR RNAs, which don’t have pre-existing 3’-OH ends. Pre-adenylated adapters are then ligated using T4 RNA ligase 1 in the absence of ATP and with a high concentration of polyethylene glycol (PEG). The 3’ capture step enables precise determination of the 3’ ends of diverse RNA molecules. Additionally, a random hexamer in the ligated ... [摘要]  新一代高通量测序技术能够对细胞中的RNA群体进行敏感和明确的分析。在这里,我们描述了一种从细菌中分离和链特异性测序小RNA池的方法,所述细菌可以在单个实验中多路复用以容纳多个生物样品。小RNA通过聚丙烯酰胺凝胶电泳分离并用T4多核苷酸激酶处理。这允许3'衔接头连接至CRISPR RNA,其不具有预先存在的3'-OH末端。然后使用T4 RNA连接酶1在不存在ATP和高浓度聚乙二醇(PEG)的情况下将前腺苷酸化的衔接子连接。 3'捕获步骤能够精确测定不同RNA分子的3'末端。此外,连接适配器中的随机六聚体有助于控制潜在的下游扩增偏差。逆转录后,将cDNA产物环化并通过PCR制备文库。我们显示扩增的文库不需要通过凝胶电泳可见,以期望产物的有效测序。使用这种方法,我们通常从少量纯化的小RNA制备RNA测序文库。该协议适合于通过对成熟的CRISPR RNA进行测序来测定细菌中的CRISPR RNA生物合成,但可以用于测序不同类型的小RNA。我们还提供了一个完整的数据处理管道示例,并提供了运行所提供脚本的说明。


【背景】与聚集的经常散布的短回文重复序列(CRISPR)相关的遗传模块赋予不同的原核宿主适应性免疫(Barrangou et ...

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 Model of Dextran Sodium Sulfate (DSS)-induced Colitis
Author:
Date:
2017-08-20
[Abstract]  Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the intestinal tract and is primarily comprised of Crohn’s disease (CD) and ulcerative colitis (UC). Several murine models that include both chemical induced and genetic models have been developed that mimic some aspects of either CD or UC. These models have been instrumental in our understanding of IBD. Of the chemical induced colitis models, dextran sodium sulfate (DSS) induced colitis model is a relatively simple and very widely used model of experimental colitis. [摘要]  炎症性肠病(IBD)是肠道的慢性炎性疾病,主要由克罗恩病(CD)和溃疡性结肠炎(UC)组成。 已经开发了几种包含化学诱导和遗传模型的鼠模型,其模拟CD或UC的某些方面。 这些模型在我们对IBD的理解方面发挥了重要作用。 在化学诱导的结肠炎模型中,葡聚糖硫酸钠(DSS)诱导的结肠炎模型是相对简单且非常广泛使用的实验性结肠炎模型。
【背景】炎症性肠病(IBD)是一种复杂而多因素的疾病,病因不明(Sartor,2006)。然而,多种因素被认为是赋予IBD易感性的关键,例如宿主遗传学,环境触发,对微生物和膳食抗原的异常免疫应答的缺陷(Sartor,2006)。可以提供几种针对疾病过程特定方面的小鼠模型(Mizoguchi,2012)。 DSS诱导的实验性结肠炎是一种快速和广泛使用的肠道炎症模型(Perse和Cerar,2012)。虽然DSS诱导的结肠炎的确切机制还不太清楚,但广泛接受的是上皮单层的破坏导致潜在的免疫系统暴露于含有微生物和微生物产物的肠内容物(Perse和Cerar,2012)。 DSS诱导的结肠炎症可以通过改变饮用水中DSS的浓度,持续时间和周期来适应肠道炎症的急性,慢性或复发模型。在本协议中,我们提供了程序的详细描述,执行协议时的重要注意事项。我们已经使用这个协议来解决我们以前的工作中Muc4在DSS诱导性结肠炎中的作用(Das et al。,2015)。

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