Name: identification of circular rnas using rna sequencing
Uploaded: 2019-11-14T11:00:00
Description: Watch this Scientific Journal Video about Identification of Circular RNAs using RNA Sequencing at JoVE.com

我们感谢亚利桑那州太阳城的旗帜太阳健康研究所脑和身体捐赠计划（BBDP）提供人脑组织。BBDP得到了国家神经疾病和中风研究所（U24 NS072026帕金森病及相关疾病国家脑和组织资源）、国家老龄问题研究所（P30AG19610亚利桑那州阿尔茨海默病核心中心）、亚利桑那州卫生服务部（合同211002， 亚利桑那阿尔茨海默氏症研究中心），亚利桑那生物医学研究委员会（合同4001，0011，05-901和1001到亚利桑那州帕金森病联合会）和迈克尔J。福克斯帕金森研究基金会27日。这项研究也得到了国土安全部和亚利桑那州（ADHS补助金+ADHS14-052688）的支持。我们还感谢安德里亚·施密特（班纳研究）和辛西娅·莱丘加（TGen）提供的行政支持。

这项研究是按照所有机构、国家和国际人类福利准则进行的。脑组织从位于亚利桑那州太阳城的横幅太阳健康研究所脑和身体捐献项目获得。大脑和身体捐赠计划的运作由西方机构审查委员会（WIRB协议#20120821批准）。所有主体或其法律代表在知情同意书上签了字。商业（非脑）生物标本是从蛋白酶中购买的。
1. RNase R 治疗
注：在以下步骤中，...

<ol>
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在这项研究中，测试了两个市售的库制备试剂盒、预处理选项和输入RNA量，以优化circRNA扩充协议，用于构建circRNA测序库。根据本研究的评估，在创建 circRNA 测序库方面，一些关键方面和关键步骤是显而易见的。我们的评估证实了RNase R预处理的效用，这反映在检测到的环RNA数量的增加上。总体而言，在使用具有RNase R预处理和4μg输入RNA的Illumina TruSeq库试剂盒时，环RNA的倍增性更高。这些结果与先?...

循环RNA（CircRNA）是内源性的、非编码的RNA，由于它们在真核转录组1、2、3中的普遍表达而备受关注。它们形成于外向的回拼接时，因此最初被认为是拼接伪像4，5。然而，最近的研究表明，circRNA表现出细胞类型，组织和发育阶段的具体表达3，6和进化保存2，3。此外，它们还参与调节蛋白质-蛋白质相互作用7、微RNA（miRNA）结合3、8、9、10和调节亲基因转录11。
在经典RNA测序（RNA-seq）中，由于多A选择mRNA，在库构建过程中，循环RNA可能完全丢失，或者由于丰度低，可能难以分离。然而，最近的circRNA表征研究已经纳入了使用RNase R的预处理步骤，以丰富circRNA2，12，13。RNase R 是一种驱外酶，可消化线性RNA，留下圆形RNA结构。CircRNA扩充方案通过生成和比较来自两个市售的全转录组构建试剂盒的数据进行优化，无论是否采用RNase R预处理步骤，并使用不同量的总RNA输入（1至4μg）。接下来，优化的协议用于评估五个不同大脑区域（小脑 [BC]、下垂叶 [IP]、中端脑环叶 [MG]、下垂皮层 [OC] 和上等前额陀螺 [SF]） 和四个其他组织类型（肝脏 [LV]、肺 [LU]、淋巴结 [LN] 和胰腺] 的环状RNA的丰度。RNA-seq库对端测序，并使用六种不同的circRNA预测算法对数据进行分析：find_circ3，CIRI14，Mapsplice15，KNIFE16，DCC17和CIRCexplorer18。根据我们的分析，当使用带有RNase R预处理和4μg总输入RNA的搁浅总RNA库制备试剂盒时，检测到的唯一循环RNA数量最多。此处介绍了优化的协议。正如先前报道的19，20，与其他组织类型相比，大脑中观察到的环RNA的富集性最高。

<table>
	<tbody>
		<tr>
			<td>1000 &#181;L pipette tips</td>
			<td>Rainin</td>
			<td>GP-L1000F</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#181;L pipette tips</td>
			<td>Rainin</td>
			<td>SR L 10F</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L pipette tips</td>
			<td>Rainin</td>
			<td>SR L 200F</td>
			<td></td>
		</tr>
		<tr>
			<td>2200 TapeStation Accessories (foil covers)</td>
			<td>Agilent Technologies</td>
			<td>5067-5154</td>
			<td></td>
		</tr>
		<tr>
			<td>2200 TapeStation Accessories (tips)</td>
			<td>Agilent Technologies</td>
			<td>5067-5153</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive Film for Microplates</td>
			<td>VWR</td>
			<td>60941-064</td>
			<td></td>
		</tr>
		<tr>
			<td>AMPure XP Beads 450 mL</td>
			<td>Beckman Coulter</td>
			<td>A63882</td>
			<td>PCR purification</td>
		</tr>
		<tr>
			<td>Eppendorf twin.tec 96-Well PCR Plates</td>
			<td>VWR</td>
			<td>951020401</td>
			<td></td>
		</tr>
		<tr>
			<td>High Sensitivity D1000 reagents</td>
			<td>Agilent Technologies</td>
			<td>5067-5585</td>
			<td></td>
		</tr>
		<tr>
			<td>High Sensitivity D1000 ScreenTape</td>
			<td>Agilent Technologies</td>
			<td>5067-5584</td>
			<td></td>
		</tr>
		<tr>
			<td>HiSeq 2500 Sequencing System</td>
			<td>Illumina</td>
			<td>SY-401-2501</td>
			<td></td>
		</tr>
		<tr>
			<td>HiSeq 3000/4000 PE Cluster Kit</td>
			<td>Illumina</td>
			<td>PE-410-1001</td>
			<td></td>
		</tr>
		<tr>
			<td>HiSeq 3000/4000 SBS Kit (150 cycles)</td>
			<td>Illumina</td>
			<td>FC-410-1002</td>
			<td></td>
		</tr>
		<tr>
			<td>HiSeq 4000 Sequencing System</td>
			<td>Illumina</td>
			<td>SY-401-4001</td>
			<td></td>
		</tr>
		<tr>
			<td>HiSeq PE PE Rapid Cluster Kit v2</td>
			<td>Illumina</td>
			<td>PE-402-4002</td>
			<td></td>
		</tr>
		<tr>
			<td>HiSeq Rapid SBS Kit v2 (50 cycle)</td>
			<td>Illumina</td>
			<td>FC-402-4022</td>
			<td></td>
		</tr>
		<tr>
			<td>Kapa Total RNA Kit</td>
			<td>Roche</td>
			<td>KK8400</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular biology grade ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP28184</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit Assay Tubes</td>
			<td>Supply Center by Thermo Fischer</td>
			<td>Q32856</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit dsDNA High Sense Assay Kit</td>
			<td>Supply Center by Thermo Fischer</td>
			<td>Q32854</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA cleanup and concentrator - 5</td>
			<td>Zymo</td>
			<td>RCC-100</td>
			<td>Contains purification columns, collection tubes</td>
		</tr>
		<tr>
			<td>RNAClean XP beads</td>
			<td>Beckman Coulter Genomics</td>
			<td></td>
			<td>RNA Cleanup beads</td>
		</tr>
		<tr>
			<td>Rnase R</td>
			<td>Lucigen</td>
			<td>RNR07250</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript II Reverse Transcriptase 10,000 units</td>
			<td>ThermoFisher (LifeTech)</td>
			<td>18064014</td>
			<td></td>
		</tr>
		<tr>
			<td>TapeStation 2200</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td>Nucleic Acid analyzer</td>
		</tr>
		<tr>
			<td>TElowE</td>
			<td>VWR</td>
			<td>10128-588</td>
			<td></td>
		</tr>
		<tr>
			<td>TruSeq Stranded Total RNA Library Prep Kit</td>
			<td>Illumina</td>
			<td>20020596</td>
			<td>Kit used in section 3</td>
		</tr>
		<tr>
			<td>Two-Compartment Divided Tray</td>
			<td>VWR</td>
			<td>3054-1004</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Water</td>
			<td>Supply Center by Thermo Fischer</td>
			<td>10977-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal control RNA</td>
			<td>Agilent</td>
			<td>740000</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of circular rnas using rna sequencing

循环RNA（循环RNA）是一类非编码RNA，涉及微RNA（miRNA）调节、蛋白质-蛋白质相互作用的中介和父母基因转录的调节等功能。在传统的下一代RNA测序（RNA-seq）中，在mRNA库的构建过程中，由于多A选择，循环RNA通常被忽视，或者发现丰度非常低，因此难以分离和检测。在这里，通过比较库制备试剂盒、预处理选项和各种RNA输入总量，优化了circRNA库构建协议。测试了两个市售全转录组库制备试剂盒，带和无RNase R预处理，并使用可变量的总RNA输入（1至4μg）。测试。最后，多种组织类型;包括肝、肺、淋巴结和胰腺;以及多个大脑区域;包括小脑、下垂叶、中时陀螺、腹皮层和上额前陀螺;比较以评估组织类型的环RNA丰度。使用六种不同的环RNA检测工具（find_circ、CIRI、Mapsplice、KNIFE、DCC和CIRCexplorer）对生成的RNA-seq数据进行分析后发现，具有RNase R预处理和4μgRNA输入的搁浅总RNA库制备试剂盒是最佳选择用于识别最高相对数量的环RNA 的方法。与以前的发现一致，与其他组织类型相比，在脑组织中观察到的环RNA的富集性最高。

使用市售的通用控制RNA（UC）和使用两个库制备试剂盒生成的数据，这两个试剂盒在其协议中都包括核糖核酸消耗步骤。使用分析工作流（数据分析工作流，第 4 节），总体而言，TruSeq 数据集中检测到的环RNA 数量高于 Kapa 数据集（图 1）。尽管两个试剂盒的数据集中的核糖体RNA （rRNA） 百分比低于 5%，输入量较低（1，2 微克），但 Kapa 数据集的 rRNA 含量较高，为 4、5 和 10 ug...

Watch this Scientific Journal Video about Identification of Circular RNAs using RNA Sequencing at JoVE.com

Identification of Circular RNAs using RNA Sequencing

循环RNA（循环RNA）是非编码RNA，在转录调节和蛋白质之间的中介相互作用中可能发挥作用。在对构建环RNA测序库的不同参数进行评估后，利用RNase R预处理的搁浅总RNA库制备编制了一个协议，并在此提出。

使用RNA测序识别循环RNA

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein ...

该协议是评估圆RNA库制备的关键方面的结果，如试剂盒类型、RNase R治疗、输入量及其对圆环RNA检测的影响。它可实现最佳的圆环RNA检测，并根据用户的需求提供有关可调参数的数据。识别不同样本类型的圆环RNA将有助于我们更好地了解其表达的分布，并设置这些分子的功能作用。此方法可应用于任何总RNA样本。在开始协议之前，将RNA样本放在冰上是重要的。在准备前评估安捷伦生物分析器或磁带站上的 RIN 和 DV200 值，并在使用 RNA 时遵循适当的步骤。首先在39微升无RNase水中将总RNA稀释至4微克，然后上下移液混合。在单独的管中，使用 1x RNase R 缓冲液将 RNase R 稀释到每微升两个单位的工作浓度。将总RNA的39微升和5个微升的10xRNase R反应缓冲液放入1.5微升反应管中，并混合。加入稀释的 RNase R.的六微升，然后将移液器调整为全反应量，然后通过上下移液 10 次混合溶液。将管子放在37摄氏度的水浴中10分钟，确保将全部反应量浸入水浴中。然后，将管子放在冰上，并立即进行RNA清理和浓缩。在开始之前，准备RNA洗涤缓冲液，将浓缩液与48毫升100%乙醇混合，并放入收集管中。准备就绪后，将两卷RNA结合缓冲液添加到RNase R处理样品中，并很好地混合。在混合物中加入150微升100%乙醇，共300微升，将整个体积转移到柱子上，并离心机30秒。去除流，并将400微升RNA准备缓冲液直接添加到柱子中。离心30秒，然后丢弃流经。然后，将700微升RNA洗涤缓冲液加入柱，离心机30秒，然后丢弃流经。再添加 400 微升的洗涤缓冲器，离心机两分钟，并将柱转移到新的无 RNase 1.5 毫升管中。将移液器尖端在柱过滤器正上方，小心地将 11 微升无 RNase 水直接添加到柱中。让样品在室温下坐一分钟，然后离心一分钟。确保 1.5 毫升管中有流量，然后丢弃柱。纯化的RNA样品可以储存在零下80摄氏度，或立即用于库准备。将纯化总RNA的10微升转移到96井PCR板中的清洁井中。加入五微升的 rRNA 结合缓冲液，然后加入五升 rRNA 去除混合微升，然后轻轻上下移液器混合试剂。密封板，并在68摄氏度的高温下在预编程和预热热循环器块上孵育5分钟。孵育后，让板在室温下坐一分钟。从板上取下密封件，并将 35 微升涡旋室温 rRNA 去除珠添加到样品中。将移液器调整为 45 微升，上下移液器 10 至 20 次以彻底混合。让板在室温下坐一分钟。将板转移到磁性支架上，在那里孵育一分钟或直到溶液清除。将上一液转移到板上的新井。然后将板从磁性支架转移到板支架上。涡流RNA清理珠子，直到它们被很好地分散，并添加99微升珠到样品。上下移液混合，在室温下孵育板10分钟。将板转移到磁性支架上，并在那里保留五分钟，或直到溶液清除，然后从井中清除并丢弃所有上清液。当板还在磁支架上时，在井中加入200微升新鲜准备好的80%乙醇，而不会打乱珠子。将乙醇留在井中30秒，然后取出并丢弃。向井中加入 11 微升的洗脱缓冲液，然后上下移液混合。在室温下孵育板两分钟，然后将其转移回磁性支架，并一直放在该板，直到溶液清除。将 8.5 微升的上一液转移到同一板上的新井中，并将 8.5 微升的 Elute、Primer、碎片高混合添加到每个包含样品的井中。上下移液10次，以彻底混合，密封板，并在热循环器预热至94摄氏度中孵育8分钟。将样品冷却至4摄氏度，从热循环器中取出板，并短暂离心。为该协议评估了两个图书馆准备工具包，TruSeq和KAKA。总体而言，使用TruSeq试剂盒时检测到的圆形RNA数量和核糖核糖核RNA百分比较低，因此选择TruSeq进行进一步实验。通过比较预处理和非预处理库生成的数据，评估了 RNase R 预处理的重要性。正如预期的那样，与未预处理的库相比，在预处理库中始终识别着数量较高的循环RNA。输入RNA的量经过优化，可检测循环RNA的更高多样性。库使用1、2和4微克输入RNA，使用4微克总RNA时观察到循环RNA物种的最高多样性。优化方案用于比较来自四个健康捐赠者的五个大脑区域的循环RNA丰度，以及来自六个健康捐赠者的其他组织类型。总的来说，与其他组织类型相比，大脑中观察到的圆形RNase的丰度更高。在开始协议之前，在使用 RNA 时，必须记住遵循适当的程序，包括戴手套、用 RNase 湿巾彻底清洁长凳和移液器或喷雾器，以及事先将 RNA 放在冰上。按照这个程序，可以执行正交验证，如桑格测序识别的圆RNA结。新的环RNA的发现和它们存在的进一步证据开辟了探索其生物功能的新研究领域。

Research

JoVE Journal

Genetics

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科研

遗传学

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

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Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

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的转录分析<em&gt;ç</em&gt;。<em&gt;线虫</em&gt; RNA测序数据通过炫套房的银河项目

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Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

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Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

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Rare Event Detection Using Error-corrected DNA and RNA Sequencing

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Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

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利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

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Overexpressing Long Noncoding RNAs Using Gene-activating CRISPR

使用基因激活 crispr 过度表达长非编码 rna

Long noncoding RNA (lncRNA) biology is a new and exciting field of research, with the number of publications from this field growing exponentially since ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

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