| mRNA Extraction from Gill Tissue for RNA-sequencing
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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 ...
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| Gene Expression Analysis of Sorted Cells by RNA-seq in Drosophila Intestine
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Author:
Date:
2016-12-20
[Abstract] RNA sequencing (RNA-seq) has become a popular method for profiling gene expression. Among many applications, one common purpose is to identify differentially expressed genes and pathways in different biological or pathological conditions. This protocol provides detailed procedure for RNA-seq analysis of ~250,000 sorted Drosophila intestinal cells (Chen et al., 2016), in which RNA amplification is not required.
[摘要] RNA测序（RNA-seq）已经成为描述基因表达的流行方法。 在许多应用中，一个共同目的是在不同的生物学或病理学条件下鉴定差异表达的基因和途径。 该方案提供了〜250,000个分选的果蝇肠细胞的RNA-seq分析的详细程序（Chen等，2016），其中不需要RNA扩增。
【背景】RNA-seq的转录组分析已经成为鉴定不同生物或病理条件下差异表达基因和途径的常用方法。 对于产生低mRNA水平的样品，通常在深度测序之前进行RNA或cDNA扩增（Dutta等，2015）。 然而，这个程序可能会潜在地省略以低丰度表达的重要候选人。 在这里，我们提供了不需要RNA扩增的分选的果蝇肠细胞的RNA-seq分析的详细程序。
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| ACE-score-based Analysis of Temporal miRNA Targetomes During Human Cytomegalovirus Infection Using AGO-CLIP-seq
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Author:
Date:
2016-04-20
[Abstract] Although temporal regulation of gene expression during the course of infection is known to be critical for determining the outcome of host-virus interactions, systematic temporal analysis of the miRNA targetomes during productive viral infection has been technically challenging due to the large range of miRNA-mRNA cross-talks at the host-virus interface. High-confidence quantifying models of the suppression efficacy in targeting sites by integrating bioinformatics with Argonaute-crosslinking and immunoprecipitation followed by high-throughput sequencing (AGO-CLIP-seq) (Chi et al., 2009) data have been poorly developed. To accurately identify miRNA target sites and calculate the targeting efficacy of miRNA-target interactions, we developed a new bioinformatic quantitation method, ...
[摘要] 尽管已知在感染过程中基因表达的时间调节对于确定宿主 - 病毒相互作用的结果是至关重要的，但是在生产性病毒感染期间对miRNA targetomes的系统时间分析在技术上是具有挑战性的，因为大范围的miRNA- mRNA在主机 - 病毒接口交叉对话。数据通过将生物信息学与Argonaute-交联和免疫沉淀接着高通量测序（AGO-CLIP-seq）数据（Chi等人，2009）数据结合，已经不发达。为了准确地鉴定miRNA靶位点并计算miRNA-靶相互作用的靶向效果，我们开发了新的生物信息学定量方法，即AGO-CLIP-seq富集（ACE） - 评分算法（Kim等， 2015）。在我们的AGO-CLIP-seq分析中包括未感染的对照可以显着提高病毒或人miRNA的真实靶位点识别的准确性，并且在我们的ACE评分方法中提取生产性人巨细胞病毒（HCMV）感染期间的生理学显着变化。因此，我们建议我们新的基于ACE评分的方法可以应用于各种miRNA targetome研究，这将在其他类型的时间背景下进行，如发展阶段，细胞因子或病原体的免疫刺激和其他病毒。
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