Enrichment of mRNA and Bisulfite-mRNA Library Preparation for Next-Generation Sequencing

Szu-Ying Chen; Po-Hsien Huang

doi:10.3791/65352

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Biologia

mRNA和亚硫酸氢盐-mRNA文库的富集制备用于二代测序

Published: July 07, 2023

doi:

10.3791/65352

Szu-Ying Chen², Po-Hsien Huang²

¹Institute of Basic Medical Sciences, College of Medicine,National Cheng Kung University, ²Department of Biochemistry and Molecular Biology, College of Medicine,National Cheng Kung University

Summary

该协议提供了一种易于遵循的工作流程，可使用标准化设备对感兴趣的生物样品进行poly（A）RNA纯化、亚硫酸氢盐转化和文库制备。

Abstract

各种类型的 RNA 转录本中的 RNA 转录后修饰与真核细胞中的多种 RNA 调控有关。异常的 RNA 5-甲基胞嘧啶修饰和 RNA 甲基转移酶的失调表达已被证明与包括癌症在内的多种疾病有关。开发了全转录组亚硫酸氢盐测序，以表征亚硫酸氢盐转化的 RNA 在碱基对分辨率下的位置和定量胞嘧啶甲基化水平。在此，该协议详细介绍了两轮poly（A）RNA纯化，三个亚硫酸氢盐反应循环和文库制备的程序，以允许mRNA 5-甲基胞嘧啶修饰位点的转录组范围定位。主反应后RNA数量和质量的评估对于监测RNA完整性至关重要，也是确保高质量测序文库的关键步骤。理想情况下，这些程序可以在三天内完成。通过该方案，使用高质量的总RNA作为输入，实际上可以建立强大的亚硫酸氢盐-mRNA文库，用于从目标样品进行二代测序。

Introduction

在 150 多种转录后修饰¹ 中，已在各种类型的 RNA 中鉴定出 5-甲基胞嘧啶（m⁵C）修饰，包括核糖体 RNA、转移 RNA、信使 RNA、micro RNA、长链非编码 RNA、vault RNA、增强子 RNA 和小 cajal 体特异性 RNA²。RNA m 5 C 与多种生物学和病理机制有关，例如调节植物根系发育³、病毒基因表达⁴ 和癌症进展⁵。该协议的目的是提供简化的管道，以表征处于不同发育阶段或疾病环境中的生物样品的转录组范围的mRNA m⁵C修饰谱。开发了全转录组亚硫酸氢盐测序来表征亚硫酸氢盐转化 RNA 中碱基对分辨率^{为 6}、⁷^、⁸^、⁹ 的位置和定量胞嘧啶甲基化水平。这在研究 m⁵C 与参与细胞生物调控机制的基因表达和 RNA 命运的关联时特别有用。在哺乳动物细胞中，有两种已知的 m 5 C 读取器：ALYREF 可以识别细胞核的 m 5 C 并充当 mRNA 细胞核到胞质溶胶转运蛋白¹⁰，而 YBX1 可以识别细胞质中的 m⁵C 并增加 mRNA 稳定性¹¹。系统性红斑狼疮 CD4+ T 细胞中报告了与免疫通路相关的异常 m⁵C mRNA¹²。研究表明，mRNA m⁵C 修饰与癌症免疫调节和癌症进展之间存在关联^13,14。因此，在mRNA上绘制m⁵C修饰图谱可以为阐明潜在的调控机制提供关键信息。

为了研究RNA m 5 C修饰在某些生物学条件下的功能作用，基于亚硫酸氢盐转化（bsRNA-seq）和基于抗体亲和富集的方法，如m 5 C-RIP-seq、miCLIP-seq和5-Aza-seq，可以与高通量测序平台相结合，在转录组范围内使用m⁵C修饰对靶区和序列进行高效检测¹⁵^，¹⁶.该协议的优势在于，由于基于抗体亲和富集的方法依赖于高质量抗体的可用性，因此该协议的优势在于单碱基分辨率下提供了全面的RNA m 5 C景观，并且可以实现m⁵C甲基化景观的单片段分辨率¹⁷。

所有 RNA 样品都将使用 oligo（dT）微球进行两轮 mRNA 富集、三个亚硫酸氢盐反应循环和测序文库制备。为了监测 RNA 质量，将在 mRNA 纯化和亚硫酸氢盐反应程序前后通过毛细管凝胶电泳检查每个 RNA 样品，以评估片段分布。纯化的文库将通过其 PCR 扩增子质量进行检查，通过毛细管凝胶电泳检查 DNA 大小分布片段，并在测序前通过基于荧光染料的定量测定检查其总量。该系统还可用于分析广谱生物样品，如农产品、分离的病毒粒子、细胞系、模式生物和病理标本。

Protocol

1. Poly（A） RNA纯化注：在进行poly（A） RNA纯化之前，使用用DNase I处理的总RNA，并通过毛细管或常规凝胶电泳评估检查总RNA的质量和完整性。研究人员应该能够识别高分子量场中的 28S 和 18S rRNA 核糖体条带以及低分子量场中的 5.8S rRNA 带，而电泳图中没有任何明显的拖尾带。纯化步骤基本上遵循制造商的说明，并在特定步骤中进行了细微的修改。有关本协议中使用?…

Representative Results

按照本报告中的程序从细胞系19 中生成一系列bsRNA-seq文库（图1）。在对细胞系样品进行总RNA纯化并进行DNase处理并通过凝胶电泳和紫外-可见分光光度法（A260/A280）检查质量后，RNA样品可以进行poly（A）RNA富集。为了确定双重纯化是否可以去除大部分核糖体RNA，通过毛细管电泳总RNA测定法评估poly（A） RNA的纯化效率，该测定法可以自动计算rRNA污…

Discussion

在该协议中，通过使用标准化组件实现了聚（A）富集，亚硫酸氢盐转化和文库制备的详细流程。进一步的测序分析提供了目标样品中mRNA 5-甲基胞嘧啶的鉴定。

关键步骤是起始材料（总RNA）的质量，因为RNA的降解会影响poly（A） RNA纯化的回收率。在进行poly（A） RNA纯化步骤之前，应小心处理样品并避免RNase污染。该过程的另一个关键部分是文库制备中的PCR循环次数。循环数的?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

这项工作得到了台湾国家科学技术委员会的支持。[NSTC 111-2314-B-006-003]

Materials

Agilent 2100 Electrophoresis Bioanalyzer System	Agilent, Santa Clara, CA		RNA quality detection
AMpure XP beads	Beckman Coulter	A63881	purify DNA
Bioanalyzer DNA high sensitivity kit	Agilent, Santa Clara, CA	5067-4626	DNA quality dection
Bioanalyzer RNA 6000 Pico kit	Agilent, Santa Clara, CA	5067-1513	RNA quality dection
DiaMag02 – magnetic rack	Diagenode, Denville, NJ	B04000001	assist library preparation
DiaMag1.5 – magnetic rack	Diagenode, Denville, NJ	B04000003	assist poly(A) RNA purificaion
Dynabeads mRNA DIRECT purification kit	Thermo Fisher Scientific, Waltham, MA	61011	poly(A) RNA purificaion; Wash Buffer 1 and Wash Buffer 2
Ethanol	J.T.Baker	64-17-5
EZ RNA methylation kit	Zymo, Irvine, CA	R5002	bisulfite treatment
Firefly luciferase mRNA	Promega, Madison, WI, USA	L4561	spike in control seqeunce
KAPA Library Quantification Kits	Roche, Switzerland	KK4824	library quantification
Nanodrop spectrophotometer	Thermo Fisher Scientific, Waltham, MA		Total RNA quantity detection
NEBNext multiplex Oligos for illumina (index Primer set1)	New England Biolabs, Ipswich, MA	E7335S	library preparation
NEBNext Ultra Directional RNA Library Prep Kit for Illumina	New England Biolabs, Ipswich, MA	E7760S	library preparation
Nuclease-free Water	Thermo Fisher Scientific	AM9932
P2 pipetman	Thermo Fisher Scientific, Waltham, MA	4641010
Qubit 2.0 fluorometer	Thermo Fisher Scientific, Waltham, MA		RNA quantity detection
Qubit dsDNA HS Assay Kit	Thermo Fisher Scientific, Waltham, MA	Q32854	DNA quantity detection
Qubit RNA HS Assay Kit	Thermo Fisher Scientific, Waltham, MA	Q32852	RNA quantity detection

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Chen, S., Huang, P. Enrichment of mRNA and Bisulfite-mRNA Library Preparation for Next-Generation Sequencing. J. Vis. Exp. (197), e65352, doi:10.3791/65352 (2023).