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

Published: July 7th, 2023



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

This protocol provides an easy-to-follow workflow to conduct poly(A) RNA purification, bisulfite conversion, and library preparation using standardized equipment for a biological sample of interest.

RNA post-transcriptional modifications in various types of RNA transcripts are associated with diverse RNA regulation in eukaryotic cells. Aberrant RNA 5-methylcytosine modifications and the dysregulated expression of RNA methyltransferases have been shown to be associated with various diseases, including cancers. Transcriptome-wide bisulfite-sequencing was developed to characterize the positions and the quantitative cytosine methylation levels in the bisulfite-converted RNA at the base-pair resolution. Herein, this protocol presents the procedures of two rounds of poly(A) RNA purification, three cycles of bisulfite reaction, and library preparation in detail to allow the transcriptome-wide mapping of mRNA 5-methylcytosine modification sites. The assessment of RNA quantity and quality after the main reaction is essential to monitor RNA integrity and is a critical step for ensuring high-quality sequencing libraries. Ideally, the procedures can be completed within three days. With this protocol, using high-quality total RNA as the input can practically build up robust bisulfite-mRNA libraries for next-generation sequencing from the sample of interest.

Among over 150 types of post-transcriptional modifications1, 5-methylcytosine (m5C) modification has been identified in various types of RNAs, including ribosomal RNA, transfer RNA, messenger RNA, micro RNA, long non-coding RNA, vault RNA, enhancer RNA, and small cajal body-specific RNAs2. The RNA m5C is associated with diverse biological and pathological mechanisms such as regulating plant root development3, viral gene expression4, and cancer progression5. The aim of this protocol is to provide streamlined pipelines to characte....

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1. Poly(A) RNA purification

NOTE: Use the total RNA treated with DNase I and examine the total RNA quality and integrity by capillary or conventional gel electrophoresis assessment before proceeding to poly(A) RNA purification. Investigators should be able to identify the 28S and 18S rRNA ribosomal bands in the high molecular weight field and the 5.8S rRNA band in the low molecular weight field without any significant smear bands in the electropherogram. The purification steps e.......

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A series of bsRNA-seq libraries from cell lines19 were generated by following the procedures in this report (Figure 1). After total RNA purification accompanied by DNase treatment performed on cell line samples and the quality checked by gel electrophoresis and UV-Vis spectrophotometry (A260/A280), the RNA sample can proceed to poly(A) RNA enrichment. To determine whether the double purification could remove the majority of ribosomal RNA, the purifica.......

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In this protocol, a detailed pipeline of poly(A) enrichment, bisulfite conversion, and library preparation was achieved by utilizing standardized components. Further sequencing analysis provided the identification of mRNA 5-methylcytosine in samples of interest.

The critical step is the quality of starting material-total RNA-since the degradation of RNA would impact the recovery rate of poly(A) RNA purification. The sample should be carefully handled and RNase contamination avoided before cond.......

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This work was supported by the National Science and Technology Council of Taiwan. [NSTC 111-2314-B-006-003]


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Name Company Catalog Number Comments
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 Equation 1 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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