| A Small RNA Isolation and Sequencing Protocol and Its Application to Assay CRISPR RNA Biogenesis in Bacteria
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Author:
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 ...
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| Mungbean Yellow Mosaic India Virus (MYMIV)-infection, Small RNA Library Construction and Deep Sequencing for MicroRNA Identification in Vigna mungo
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Author:
Date:
2016-10-20
[Abstract] This protocol describes small RNA library preparation from Vigna mungo total RNA followed by deep sequencing and analysis for microRNA identification.
[摘要] 这个协议描述从猕猴真菌总RNA的小RNA文库制备，然后深度测序和分析microRNA识别。
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| Quantification of Total and 2-LTR (Long terminal repeat) HIV DNA, HIV RNA and Herpesvirus DNA in PBMCs
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Author:
Date:
2015-06-05
[Abstract] Almost all individuals infected with human immunodeficiency virus (HIV) are also infected with cytomegalovirus (CMV) and Epstein Barr virus (EBV). The aims of our studies have included characterizing and measuring the latent HIV reservoir and understanding the association between asymptomatic replication of CMV (and other herpesvirus, including EBV) and this HIV reservoir (Gianella et al., 2014). This protocol was designed to simultaneously co-extract DNA and RNA from the same peripheral blood mononuclear cell (PBMC) aliquot and quantify HIV, CMV and EBV DNA, as well as HIV RNA using droplet digital PCR (ddPCR).
For collection and processing of male genital secretions and quantification of HIV RNA and DNA from seven human herpesviruses from seminal plasma, refer to ...
[摘要] 几乎所有感染人免疫缺陷病毒（HIV）的个体也被巨细胞病毒（CMV）和EB病毒（EBV）感染。 我们的研究的目的包括表征和测量潜伏的HIV库并且理解CMV（和其他疱疹病毒，包括EBV）的无症状复制与该HIV库之间的关联（Gianella等人，2014） 。 该方案设计为从相同的外周血单核细胞（PBMC）等分试样同时共提取DNA和RNA，并使用液滴数字PCR（ddPCR）定量HIV，CMV和EBV DNA以及HIV RNA。
关于男性生殖器分泌物的收集和处理以及来自七种人疱疹病毒的HIV RNA和DNA的定量，参见方案"Vacs RNA和人疱疹病毒DNA在Seminal血浆中的定量（Vargas-Meneses等人，2015）。
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