DigiTAG–a RNA Sequencing Approach to Analyze Transcriptomes of Rare Cell Populations in Drosophila melanogaster
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Author:
Date:
2020-11-05
[Abstract] Cell-type specific transcriptional programs underlie the development and maintenance of organs. Not only distinct cell types within a tissue, even cells with supposedly identical cell fates show a high degree of transcriptional heterogeneity. Inevitable, low cell numbers are a major hurdle to study transcriptomes of pure cell populations. Here we describe DigiTAG, a high-throughput method that combines transposase fragmentation and molecular barcoding to retrieve high quality transcriptome data of rare cell types in Drosophila melanogaster. The protocol showcases how DigiTAG can be used to analyse the transcriptome of rare neural stem cells (type II neuroblasts) of Drosophila larval brains, but can also be utilized for other cell types or model systems.
[摘要] [摘要]细胞类型的特定转录程序是器官的发育和维持的基础。不仅组织内不同的细胞类型,甚至具有相同细胞命运的细胞也显示出高度的转录异质性。不可避免的是,低细胞数量是研究纯细胞群体转录组的主要障碍。在这里,我们介绍DigiTAG ,这是一种高通量方法,将转座酶片段化和分子条形码相结合,以检索果蝇中稀有细胞类型的高质量转录组数据。该协议展示了DigiTAG如何可用于分析果蝇幼虫的罕见神经干细胞(II型成神经细胞)的转录组 大脑,但也可以用于其他细胞类型或模型系统。
[背景]在发育过程中,不同细胞类型之间的过渡与组织稳态之间的关系是由大量转录因子及其诱导的转录变化所精心安排的。在过去的十年中,RNA测序(RNA- seq )已成为测量整个基因组转录动力学的经典方法(Stark等,2019)。组织上的大量RNA序列不允许研究不同细胞群体的转录网络,特别是稀有细胞类型的转录网络。因此,需要提供低输入样品高质量转录组的RNA- seq方案。
在果蝇中,有限的材料通常构成分析特定组织或细胞类型的障碍。果蝇神经干细胞(称为神经母细胞)很好地说明了这一点(Homem和Knoblich ,2012)。存在成神经细胞的几个不同的亚群。例如,在果蝇的幼虫大脑中,只有16种II型成神经细胞产生神经元,神经元支配了运动和感觉处理所需的大脑区域(Walsh and ...
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Detection and Analysis of Circular RNAs by RT-PCR
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Author:
Date:
2018-03-20
[Abstract] Gene expression in eukaryotic cells is tightly regulated at the transcriptional and posttranscriptional levels. Posttranscriptional processes, including pre-mRNA splicing, mRNA export, mRNA turnover, and mRNA translation, are controlled by RNA-binding proteins (RBPs) and noncoding (nc)RNAs. The vast family of ncRNAs comprises diverse regulatory RNAs, such as microRNAs and long noncoding (lnc)RNAs, but also the poorly explored class of circular (circ)RNAs. Although first discovered more than three decades ago by electron microscopy, only the advent of high-throughput RNA-sequencing (RNA-seq) and the development of innovative bioinformatic pipelines have begun to allow the systematic identification of circRNAs (Szabo and Salzman, 2016; Panda et al., 2017b; Panda et al., ...
[摘要] 真核细胞中的基因表达在转录和转录后水平受到严格调控。 mRNA转录,mRNA转录和mRNA翻译等后转录过程由RNA结合蛋白(RBPs)和非编码(nc)RNAs控制。大量的ncRNA家族包含多种调控RNA,如microRNAs和长的非编码(lnc)RNAs,但也是探索不足的一类环状RNAs。虽然三十多年前电子显微镜首次发现,但只有高通量RNA测序(RNA-seq)的出现和创新生物信息学管道的开发已经开始允许系统鉴定circRNA(Szabo和Salzman,2016;熊猫,2017b;熊猫等,2017c)。然而,通过RNA测序鉴定的真正的circRNA的验证需要其他分子生物学技术,包括常规或定量(q)聚合酶链反应(PCR)和Northern印迹分析(Jeck和Sharpless,2014)的逆转录(RT)。使用不同引物的环状RNA的RT-qPCR分析已被广泛用于检测,验证和有时定量circRNA(Abdelmohsen等人,2015和2017; Panda等人, ,2017b)。如在此详述的,设计为跨越循环RNA后接连接序列的分歧引物可以特异性扩增circRNA而不是对应的线性RNA。总之,使用不同引物的RT-PCR分析允许直接检测和定量circRNA。
【背景】CircRNAs是共价闭合的,缺少5'或3'末端的单链RNA。虽然它们的起源知之甚少,但它们可以通过称为反向剪接的过程从前体mRNA产生(Panda等人,2017d; ...
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Polysome Fractionation to Analyze mRNA Distribution Profiles
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Author:
Date:
2017-02-05
[Abstract] Eukaryotic cells adapt to changes in external or internal signals by precisely modulating the expression of specific gene products. The expression of protein-coding genes is controlled at the transcriptional and post-transcriptional levels. Among the latter steps, the regulation of translation is particularly important in cellular processes that require rapid changes in protein expression patterns. The translational efficiency of mRNAs is altered by RNA-binding proteins (RBPs) and noncoding (nc)RNAs such as microRNAs (Panda et al., 2014a and 2014b; Abdelmohsen et al., 2014). The impact of factors that regulate selective mRNA translation is a critical question in RNA biology. Polyribosome (polysome) fractionation analysis is a powerful method to assess the association of ...
[摘要] 真核细胞通过精确调节特定基因产物的表达来适应外部或内部信号的变化。蛋白质编码基因的表达受到转录和转录后水平的控制。在后面的步骤中,翻译的调节在需要蛋白质表达模式快速变化的细胞过程中特别重要。 mRNA的翻译效率由RNA结合蛋白(RBP)和非编码(nc)RNA如微RNA(Panda等人,2014a和2014b; Abdelmohsen等人)改变2014)。调节选择性mRNA翻译的因素的影响是RNA生物学中的一个关键问题。多核糖体(多核糖体)分馏分析是评估核糖体与给定mRNA的关联的有效方法。它提供了关于该mRNA的翻译状态的有价值的信息,这取决于与它们相关联的核糖体的数目,并且鉴定未翻译的mRNA(Panda等人,2016)。与许多核糖体相关的mRNA形成大量的多核糖体,预计将被主动翻译,而与少数或没有核糖体相关的mRNA有可能翻译不佳。总之,多聚糖分馏分析允许直接测定整个转录组水平的翻译效率以及个体mRNA。
背景 ...
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