使用RNA测序识别循环RNA
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 1000 µL pipette tips | Rainin | GP-L1000F | |
| 20 µL pipette tips | Rainin | SR L 10F | |
| 200 µL pipette tips | Rainin | SR L 200F | |
| 2200 TapeStation Accessories (foil covers) | Agilent Technologies | 5067-5154 | |
| 2200 TapeStation Accessories (tips) | Agilent Technologies | 5067-5153 | |
| Adhesive Film for Microplates | VWR | 60941-064 | |
| AMPure XP Beads 450 mL | Beckman Coulter | A63882 | PCR purification |
| Eppendorf twin.tec 96-Well PCR Plates | VWR | 951020401 | |
| High Sensitivity D1000 reagents | Agilent Technologies | 5067-5585 | |
| High Sensitivity D1000 ScreenTape | Agilent Technologies | 5067-5584 | |
| HiSeq 2500 Sequencing System | Illumina | SY-401-2501 | |
| HiSeq 3000/4000 PE Cluster Kit | Illumina | PE-410-1001 | |
| HiSeq 3000/4000 SBS Kit (150 cycles) | Illumina | FC-410-1002 | |
| HiSeq 4000 Sequencing System | Illumina | SY-401-4001 | |
| HiSeq PE PE Rapid Cluster Kit v2 | Illumina | PE-402-4002 | |
| HiSeq Rapid SBS Kit v2 (50 cycle) | Illumina | FC-402-4022 | |
| Kapa Total RNA Kit | Roche | KK8400 | |
| Molecular biology grade ethanol | Fisher Scientific | BP28184 | |
| Qubit Assay Tubes | Supply Center by Thermo Fischer | Q32856 | |
| Qubit dsDNA High Sense Assay Kit | Supply Center by Thermo Fischer | Q32854 | |
| RNA cleanup and concentrator – 5 | Zymo | RCC-100 | Contains purification columns, collection tubes |
| RNAClean XP beads | Beckman Coulter Genomics | RNA Cleanup beads | |
| Rnase R | Lucigen | RNR07250 | |
| SuperScript II Reverse Transcriptase 10,000 units | ThermoFisher (LifeTech) | 18064014 | |
| TapeStation 2200 | Agilent Technologies | Nucleic Acid analyzer | |
| TElowE | VWR | 10128-588 | |
| TruSeq Stranded Total RNA Library Prep Kit | Illumina | 20020596 | Kit used in section 3 |
| Two-Compartment Divided Tray | VWR | 3054-1004 | |
| UltraPure Water | Supply Center by Thermo Fischer | 10977-015 | |
| Universal control RNA | Agilent | 740000 |
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