| 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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| Circular RT-PCR Assay Using Arabidopsis Samples
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Author:
Date:
2015-07-20
[Abstract] Post-transcriptional processing is critical for RNA biogenesis, in which conventional functional RNA transcripts are generated, such as messenger RNAs (mRNAs), transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) for translation as well as emerging non-coding RNAs with known or unknown regulatory functions. To determine the precise termini of an RNA molecule during or after processing, the primer extension and Rapid Amplification of cDNA Ends (RACE) methods have been routinely utilized for the precise mapping of 5’ or 3’ ends. Different from these assays, which are designed to detect only one end of a specific target RNA at a time, circular Reverse Transcription-Polymerase Chain Reaction (cRT-PCR) is able to simultaneously determine both the 5’ and 3’ ends of the target RNA. In Arabidopsis ...
[摘要] 转录后加工对于RNA生物发生至关重要，其中产生常规功能性RNA转录物，例如用于翻译的信使RNA（mRNA），转移RNA（tRNA）和核糖体RNA（rRNA）以及已知的新编码RNA或未知的调节功能。为了在加工期间或之后确定RNA分子的精确末端，引物延伸和cDNA末端的快速扩增（RACE）方法已经常规地用于5'或3'末端的精确作图。与设计为一次仅检测特定靶RNA的一端的这些测定法不同，环状逆转录 - 聚合酶链式反应（cRT-PCR）能够同时测定靶标的5'和3'末端RNA。在拟南芥中，cRT-PCR已经被广泛应用于鉴定核糖体RNA前体的5'和3'末端，或者评估在3'末端的长度或转录后延伸成熟mRNA。在该方案中，我们总结并提出了在拟南芥中cRT-PCR测定的详细程序，其也成功地用于我们以前公开的工作中（Hang等人 ，2014）。
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| Preparation of cDNA Library for dRNA-seq
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Author:
Date:
2012-12-05
[Abstract] microRNAs (miRNAs) are ubiquitous regulators of gene expression in eukaryotic organisms, which guide Argonaute proteins (AGO) to cleave target mRNA or inhibit its translation based on sequence complementarity. In plants, miRNA directed cleavage occurs on the target mRNA at about 10 to 11 nucleotide (nt) up stream to the site where the 5’ end of miRNA binds. Sequencing of the miRNA directed cleavage products (degradome) is widely employed as a way to both verify bioinformatic predictions of miRNA mediated regulation and identify novel targets of known miRNAs. Here we describe a protocol for preparation of degradome RNA complementary DNA library for high-through-put sequencing (dRNA-seq) using Illumina GA II sequencing platform, which is currently most popular and cost-effective. Using this ...
[摘要] 微小RNA（miRNA）是真核生物体中基因表达的普遍存在的调节剂，其指导Argonaute蛋白（AGO）基于序列互补性切割靶mRNA或抑制其翻译。在植物中，miRNA定向切割发生在靶mRNA上，在约5至11个核苷酸（nt）上游至miRNA的5'端结合的位点。 miRNA定向切割产物（降解组）的测序广泛用作验证miRNA介导的调节的生物信息学预测和鉴定已知miRNA的新靶标的方法。在这里，我们描述了使用Illumina GA II测序平台制备高通量测序（dRNA-seq）的降解组RNA互补DNA文库的方案，目前这是最受欢迎和成本效益的。使用这个协议，我们成功地使用三个茄科植物，包括烟草，番茄和马铃薯生成三个dRNA seq图书馆。尽管该协议是用单链适配器开发的，但应该能够通过用多路复用兼容的3'衔接子代替3'衔接子并用索引引物代替PCR引物来产生多重文库。
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