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DynaMag-2 Magnet

DynaMag TM -2 Magnet

Company: Thermo Fisher Scientific
Catalog#: 12321D
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Whole-genome Identification of Transcriptional Start Sites by Differential RNA-seq in Bacteria
Author:
Date:
2020-09-20
[Abstract]  Gene transcription in bacteria often starts some nucleotides upstream of the start codon. Identifying the specific Transcriptional Start Site (TSS) is essential for genetic manipulation, as in many cases upstream of the start codon there are sequence elements that are involved in gene expression regulation. Taken into account the classical gene structure, we are able to identify two kinds of transcriptional start site: primary and secondary. A primary transcriptional start site is located some nucleotides upstream of the translational start site, while a secondary transcriptional start site is located within the gene encoding sequence.

Here, we present a step by step protocol for genome-wide transcriptional start sites determination by differential RNA-sequencing (dRNA-seq) ...
[摘要]  [摘要] 细菌中的基因转录通常起始于起始密码子上游的一些核苷酸。识别SPE cific Ť ranscriptional 小号挞小号ITE (TSS)为遗传操作必需的,因为在许多情况下,起始密码子上游有中涉及的基因表达调控序列元件。考虑到经典的基因结构,我们能够鉴定出两种转录起始位点:一级和二级。主要转录起始位点位于翻译起始位点上游的一些核苷酸上,而次要转录起始位点位于基因编码序列内。

这里,我们提出一步步协议全基因组吨ranscriptional 小号馅饼小号ITES d etermination通过差RNA测序(DRNA 使用肠道病原体-SEQ)福氏痢疾杆菌血清型菌株5A作为M90T模型。但是,该方法可以用于选择的任何其他细菌物种。第一步,使用热酚法从细菌培养物中纯化总RNA。核糖体RNA(rRNA)是使用商业试剂盒通过杂交探针特异性去除的。然后准备一个富含5'- 一磷酸依赖性核酸外切酶(TEX)处理的,富含初级转录本的RNA文库,用于与未进行TEX处理的文库进行比较,然后连接已知序列的RNA接头衔接子,从而确定具有单核苷酸精度的TSS。最后,对RNA进行处理以制备Illumina测序文库,并按购买的服务进行测序。通过内部生物信息学分析鉴定TSS。

...

mRNA Extraction from Gill Tissue for RNA-sequencing
Author:
Date:
2020-03-05
[Abstract]  Adaptation is thought to proceed in part through spatial and temporal changes in gene expression. Fish species such as the threespine stickleback are powerful vertebrate models to study the genetic architecture of adaptive changes in gene expression since divergent adaptation to different environments is common, they are abundant and easy to study in the wild and lab, and have well-established genetic and genomic resources. Fish gills, due to their respiratory and osmoregulatory roles, show many physiological adaptations to local water chemistry, including differences in gene expression. However, obtaining high-quality RNA using popular column-based extraction methods can be challenging from small tissue samples high in cartilage and bone such as fish gills. Here, we describe a bead-based ... [摘要]  [摘要 ] 适应被认为是部分通过基因表达的时空变化进行的。鱼种如 三脊棘背由于对不同环境的适应性差异很普遍,因此它们是研究基因表达适应性变化的遗传结构的强大脊椎动物模型,它们在野外和实验室中丰富且易于研究,并且具有完善的遗传和基因组资源。鱼g由于其呼吸和渗透调节作用,对局部水化学表现出许多生理适应性,包括基因表达的差异。但是,从流行于软骨和骨骼的小组织样本(例如鱼g)中,使用流行的基于柱的提取方法获得高质量的RNA可能具有挑战性。在这里,我们描述了不使用纯化柱的基于珠子的mRNA提取和转录组RNA-seq协议。为了使用动物或植物组织进行各种基因表达实验,可以根据样品量轻松调整实验方案。

[背景 ] 转录组测序(RNA-seq)用于量化基因的表达水平,鉴定样品组之间基因表达水平的差异并推断基因共表达。在进化遗传学研究,RNA-SEQ可以用来作为一种方法来研究自适应发散的分子基础(例如,Rougeux 等人;,2019 Verta酒店和Jones,2019) ,鉴定候选基因底层自适应的表型(例如,费雷拉等等人,2017),并推断未知基因的功能(例如,Rawat ...

Selective Isolation of Retroviruses from Extracellular Vesicles by Intact Virion Immunoprecipitation
Author:
Date:
2018-09-05
[Abstract]  There exists a wide variety of techniques to isolate and purify viral particles from cell culture supernatants. However, these techniques vary greatly in ease of use, purity, yield and impact on viral structural integrity. Most importantly, it is becoming evident that secreted extracellular vesicles (EVs) co-purify with retroviruses using nearly all purification methods due to nearly indistinguishable biophysical characteristics such as size, buoyant density and nucleic acid content. Recently, our group has illustrated a means of isolating intact and highly enriched retroviral virions from EV-containing cell supernatants using an immunoprecipitation approach targeting the viral envelope glycoprotein of the Moloney Murine Leukemia Virus (Renner et al., 2018). This technique, that ... [摘要]  存在多种从细胞培养上清液中分离和纯化病毒颗粒的技术。然而,这些技术在易用性,纯度,产量和对病毒结构完整性的影响方面差异很大。最重要的是,由于几乎无法区分的生物物理特征,例如大小,浮力密度和核酸含量,使用几乎所有纯化方法分泌的细胞外囊泡(EV)与逆转录病毒共同纯化变得明显。最近,我们小组已经阐明了一种利用针对Moloney鼠白血病病毒的病毒包膜糖蛋白的免疫沉淀方法从含有EV的细胞上清液中分离完整和高度富集的逆转录病毒病毒颗粒的方法(Renner et al。, 2018)。这种技术,我们称之为完整的病毒粒子免疫沉淀(IVIP),使我们能够表征这些逆转录病毒表面上表位的可及性,并评估病毒包膜中病毒编码的整合膜蛋白Glycogag(gPr80)的方向。该方案的正确实施使得能够快速,简单且可重复地制备完整且高度纯化的逆转录病毒颗粒,其没有可检测的EV污染物。

【背景】广泛使用的分离逆转录病毒的方法,如人类免疫缺陷病毒(HIV)和小鼠白血病病毒(MLV),包括沉淀,色谱,超滤,超速离心,以及各种其他粒子分离方法(评论在Nestola et al。,2015)。虽然每种技术都有其特定的优点,缺点和局限性,但所有方法的共同关注点是细胞分泌的细胞外囊泡(EV)的共同纯化。

EV构成由所有细胞类型分泌的膜衍生囊泡的异质群体(Yanez-Mo ...

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