Smar2C2: A Simple and Efficient Protocol for the Identification of Transcription Start Sites
Smar2C2: A Simple and Efficient Protocol for the Identification of Transcription Start Sites
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Smar2C2：一种简单有效的转录起始位点识别方案
DOI：
10.1002/cpz1.705
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Schmitz, Robert J.
中科院分区：
文献类型：
--
作者：
Murray, Andrew;Vollmers, Christopher;Schmitz, Robert J.
启动子和驱动其功能的非编码序列是基因的基本方面，对它们的调控至关重要。转录预起始复合体结合并组装在启动子上，在那里它促进转录。转录起始点(TSS)位于启动子序列的下游，被定义为基因组中聚合酶开始将DNA转录成RNA的位置。了解TSSs的位置对于基因的注释、识别对基因调控很重要的非编码序列、检测替代TSSs以及了解5‘非编码区的含量都是有用的。几种现有的技术使得能够准确地识别TSS，但通常难以在实验上执行，需要大量的输入RNA，或者无法从单个样本中识别大量TSS。其中许多协议利用模板切换逆转录酶(TSRT)，它可靠地将接头放置在第一链合成的5‘端。在这里，我们介绍了一种利用TSRT活性结合滚环扩增来识别SSS的方法，与现有方法相比具有几个独特的优势。序列接头被放置在转录本的全长cDNA拷贝的5‘和3’端。然后，使用与这些接头兼容的夹板来环化全长cdna。通过滚环扩增得到含有串联序列的线状DNA，并对串联序列进行片段化，形成测序文库。这个方案执行起来很简单，需要相对稳定的试剂，需要有限的实验时间。使用极少量的RNA输入，该协议产生了大量准确的、全基因组范围内重复的TSS。©2023作者。基本方案1：夹板生成基本方案2：RNA提取基本方案3：cDNA合成基本方案4：DNA环化和扩增基本方案5：文库生成
Promoters and the noncoding sequences that drive their function are fundamental aspects of genes that are critical to their regulation. The transcription preinitiation complex binds and assembles on promoters where it facilitates transcription. The transcription start site (TSS) is located downstream of the promoter sequence and is defined as the location in the genome where polymerase begins transcribing DNA into RNA. Knowing the location of TSSs is useful for annotation of genes, identification of non‐coding sequences important to gene regulation, detection of alternative TSSs, and understanding of 5′ UTR content. Several existing techniques make it possible to accurately identify TSSs, but are often difficult to perform experimentally, require large amounts of input RNA, or are unable to identify a large number of TSSs from a single sample. Many of these protocols take advantage of template switching reverse transcriptases (TSRTs), which reliably place an adaptor at the 5′ end of a first strand synthesis of cDNA. Here, we introduce a protocol that exploits TSRT activity combined with rolling circle amplification to identify TSSs with several unique advantages over existing methods. Sequence adaptors are placed on the 5′ and 3′ end of the full‐length cDNA copy of a transcript. A splint compatible with those adaptors is then used to circularize the full‐length cDNA. Linear DNA containing concatemers of the cDNA are generated using rolling circle amplification, and a sequencing library is formed by fragmenting the concatemers. This protocol is straightforward to execute, requiring limited bench time with relatively stable reagents. Using extremely low amounts of RNA input, this protocol produces large numbers of accurate, deduplicated TSSs genome wide. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Splint generationBasic Protocol 2: RNA extractionBasic Protocol 3: cDNA synthesisBasic Protocol 4: cDNA circularization and amplificationBasic Protocol 5: Library generation
影响因子：
7.2
作者：
Murray, Andrew;Mendieta, John Pablo;Vollmers, Chris;Schmitz, Robert J.
通讯作者：
Schmitz, Robert J.
影响因子：
11.4
作者：
Kulpa, D;Topping, R;Telesnitsky, A
通讯作者：
Telesnitsky, A