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

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...

Creating Robust Bisulfite-mRNA Libraries for Transcriptome-Wide Mapping

<ol>
	<li>Boccaletto, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MODOMICS:+a+database+of+RNA+modification+pathways.+2021+update.">MODOMICS: a database of RNA modification pathways. 2021 update.</a> Nucleic Acids Research. 50, D231-D235 (2022).</li><li>Bohnsack, K. E., H&#246;bartner, C., Bohnsack, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eukaryotic+5-methylcytosine+(m5C)+RNA+Methyltransferases:+Mechanisms,+Cellular+Functions,+and+Links+to+Disease.">Eukaryotic 5-methylcytosine (m5C) RNA Methyltransferases: Mechanisms, Cellular Functions, and Links to Disease.</a> Genes. 10 (2), 102 (2019).</li><li>Shinde, H., Dudhate, A., Kadam, U. S., Hong, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+methylation+in+plants:+An+overview.">RNA methylation in plants: An overview.</a> Frontiers in Plant Science. 14, 1132959 (2023).</li><li>Courtney, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epitranscriptomic+addition+of+m5C+to+HIV-1+transcripts+regulates+viral+gene+expression.">Epitranscriptomic addition of m5C to HIV-1 transcripts regulates viral gene expression.</a> Cell Host &amp; Microbe. 26 (2), 217-227 (2019).</li><li>Jonkhout, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNA+modification+landscape+in+human+disease.">The RNA modification landscape in human disease.</a> RNA. 23 (12), 1754-1769 (2017).</li><li>Legrand, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistically+robust+methylation+calling+for+whole-transcriptome+bisulfite+sequencing+reveals+distinct+methylation+patterns+for+mouse+RNAs.">Statistically robust methylation calling for whole-transcriptome bisulfite sequencing reveals distinct methylation patterns for mouse RNAs.</a> Genome Research. 27 (9), 1589-1596 (2017).</li><li>Schaefer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+5-methylcytosine+analysis+by+bisulfite+sequencing.">RNA 5-methylcytosine analysis by bisulfite sequencing.</a> Methods in Enzymology. 560, 297-329 (2015).</li><li>Amort, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+5-methylcytosine+profiles+in+poly(A)+RNA+from+mouse+embryonic+stem+cells+and+brain.">Distinct 5-methylcytosine profiles in poly(A) RNA from mouse embryonic stem cells and brain.</a> Genome Biology. 18 (1), 1 (2017).</li><li>Schumann, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+links+between+5-methylcytosine+content+of+mRNA+and+translation.">Multiple links between 5-methylcytosine content of mRNA and translation.</a> BMC Biology. 18 (1), 40 (2020).</li><li>Yang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Methylcytosine+promotes+mRNA+export+-+NSUN2+as+the+methyltransferase+and+ALYREF+as+an+m5C+reader.">5-Methylcytosine promotes mRNA export - NSUN2 as the methyltransferase and ALYREF as an m5C reader.</a> Cell Research. 27, 606 (2017).</li><li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Methylcytosine+promotes+pathogenesis+of+bladder+cancer+through+stabilizing+mRNAs.">5-Methylcytosine promotes pathogenesis of bladder cancer through stabilizing mRNAs.</a> Nature Cell Biology. 21 (8), 978-990 (2019).</li><li>Guo, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disease+activity-associated+alteration+of+mRNA+m(5)+C+methylation+in+CD4(+)+T+cells+of+systemic+lupus+erythematosus.">Disease activity-associated alteration of mRNA m(5) C methylation in CD4(+) T cells of systemic lupus erythematosus.</a> Frontiers in Cell and Developmental Biology. 8 (5), 430 (2020).</li><li>Song, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+roles+of+RNA+m5C+modification+and+its+implications+in+cancer+immunotherapy.">Biological roles of RNA m5C modification and its implications in cancer immunotherapy.</a> Biomarker Research. 10 (1), 15 (2022).</li><li>Xue, C., Zhao, Y., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+RNA+cytosine-5+methylation:+detection,+regulatory+mechanisms,+biological+functions+and+links+to+cancer.">Advances in RNA cytosine-5 methylation: detection, regulatory mechanisms, biological functions and links to cancer.</a> Biomarker Research. 8 (1), 43 (2020).</li><li>Helm, M., Motorin, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+RNA+modifications+in+the+epitranscriptome:+predict+and+validate.">Detecting RNA modifications in the epitranscriptome: predict and validate.</a> Nature Reviews Genetics. 18 (5), 275-291 (2017).</li><li>Guo, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+mRNA+5-methylcytosine+modifications:+Detection,+effectors,+biological+functions,+and+clinical+relevance.">Advances in mRNA 5-methylcytosine modifications: Detection, effectors, biological functions, and clinical relevance.</a> Molecular Therapy - Nucleic Acids. 26, 575-593 (2021).</li><li>Saplaoura, E., Perrera, V., Colot, V., Kragler, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylated+RNA+Immunoprecipitation+Assay+to+Study+m5C+Modification+in+Arabidopsis.">Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis.</a> Journal of Visualized Experiments:JoVE. (159), e61231 (2020).</li><li>Quail, M. A., Swerdlow, H., Turner, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+protocols+for+the+illumina+genome+analyzer+sequencing+system.">Improved protocols for the illumina genome analyzer sequencing system.</a> Current Protocols in Human Genetics. , (2009).</li><li>Chen, S. -. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+bisulfite+sequencing+reveals+NSUN2-mediated+suppression+of+epithelial+differentiation+in+pancreatic+cancer.">RNA bisulfite sequencing reveals NSUN2-mediated suppression of epithelial differentiation in pancreatic cancer.</a> Oncogene. 41 (22), 3162-3176 (2022).</li><li>Squires, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Widespread+occurrence+of+5-methylcytosine+in+human+coding+and+non-coding+RNA.">Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA.</a> Nucleic Acids Research. 40 (11), 5023-5033 (2012).</li><li>Han, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+DNA+5-hydroxylmethylcytosine+and+RNA+5-methycytosine+reprogramming+during+early+human+development.">Dynamic DNA 5-hydroxylmethylcytosine and RNA 5-methycytosine reprogramming during early human development.</a> Genomics, Proteomics &amp; Bioinformatics. , (2022).</li><li>Amort, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+non-coding+RNAs+as+targets+for+cytosine+methylation.">Long non-coding RNAs as targets for cytosine methylation.</a> RNA biology. 10 (6), 1003-1008 (2013).</li><li>Sun, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+NSUN2+deficiency+on+the+mRNA+5-methylcytosine+modification+and+gene+expression+profile+in+HEK293+cells.">Effects of NSUN2 deficiency on the mRNA 5-methylcytosine modification and gene expression profile in HEK293 cells.</a> Epigenomics. 11 (4), 439-453 (2019).</li><li>Huang, T., Chen, W., Liu, J., Gu, N., Zhang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+identification+of+mRNA+5-methylcytosine+in+mammals.">Genome-wide identification of mRNA 5-methylcytosine in mammals.</a> Nature Structural and Molecular Biology. 26 (5), 380-388 (2019).</li><li>Rieder, D., Amort, T., Kugler, E., Lusser, A., Trajanoski, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=meRanTK:+methylated+RNA+analysis+ToolKit.">meRanTK: methylated RNA analysis ToolKit.</a> Bioinformatics. 32 (5), 782-785 (2015).</li><li>Kraus, A. J., Brink, B. G., Siegel, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+specific+oligo-based+depletion+of+rRNA.">Efficient and specific oligo-based depletion of rRNA.</a> Scientific Reports. 9 (1), 12281 (2019).</li><li>Edelheit, S., Schwartz, S., Mumbach, M. R., Wurtzel, O., Sorek, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome-wide+mapping+of+5-methylcytidine+RNA+modifications+in+bacteria,+archaea,+and+yeast+reveals+m(5)C+within+archaeal+mRNAs.">Transcriptome-wide mapping of 5-methylcytidine RNA modifications in bacteria, archaea, and yeast reveals m(5)C within archaeal mRNAs.</a> PLoS Genetics. 9 (5), e1003602 (2013).</li><li>Furlan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+methods+for+RNA+modification+detection+from+nanopore+direct+RNA+sequencing+data.">Computational methods for RNA modification detection from nanopore direct RNA sequencing data.</a> RNA Biology. 18, 31-40 (2021).</li><li>Leger, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+modifications+detection+by+comparative+Nanopore+direct+RNA+sequencing.">RNA modifications detection by comparative Nanopore direct RNA sequencing.</a> Nature Communications. 12 (1), 7198 (2021).</li><li>Mateos, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+m6A+and+m5C+RNA+modifications+at+single-molecule+resolution+from+Nanopore+sequencing.">Identification of m6A and m5C RNA modifications at single-molecule resolution from Nanopore sequencing.</a> bioRxiv. , (2022).</li><li>Johnson, Z., Xu, X., Pacholec, C., Xie, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+evaluation+of+parameters+in+RNA+bisulfite+sequencing+data+generation+and+analysis.">Systematic evaluation of parameters in RNA bisulfite sequencing data generation and analysis.</a> NAR Genomics and Bioinformatics. 4 (2), 045 (2022).</li><li>Zhang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+calibration+of+epitranscriptomic+maps+using+a+synthetic+modification-free+RNA+library.">Systematic calibration of epitranscriptomic maps using a synthetic modification-free RNA library.</a> Nature Methods. 18 (10), 1213-1222 (2021).</li><li>Chao, H. -. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+evaluation+of+RNA-Seq+preparation+protocol+performance.">Systematic evaluation of RNA-Seq preparation protocol performance.</a> BMC Genomics. 20 (1), 571 (2019).</li></ol>

The authors have no conflicts of interest to disclose.

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...

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 characterize the transcriptome-wide mRNA m5C modification profile of biological samples in different developmental stages or in the disease setting. Transcriptome-wide bisulfite-sequencing was developed to characterize the positions and the quantitative cytosine methylation levels in the bisulfite-converted RNA at base-pair resolution6,7,8,9. This is particularly useful when studying the association of m5C with gene expression and RNA fate that is involved in the biological regulatory mechanisms in cells. In the mammalian cell, there are two known m5C readers: ALYREF can recognize m5C at the nucleus and serves as an mRNA nucleus-to-cytosol transporter10, while YBX1 can recognize m5C in the cytoplasm and increase mRNA stabilization11. Aberrant m5C mRNAs related to immune pathways were reported in Systemic Lupus Erythematosus CD4+ T cells12. Studies have revealed an association between mRNA m5C modification and modulation of cancer immunity and cancer progression13,14. Hence, mapping the m5C modification profile on the mRNA can provide crucial information to elucidate the potential regulatory machinery.
To investigate the functional roles of RNA m5C modification under certain biological conditions, the bisulfite conversion-based (bsRNA-seq) and antibody affinity enrichment-based approaches such as m5C-RIP-seq, miCLIP-seq, and 5-Aza-seq can be combined with the high-throughput sequencing platform to provide efficient detection of targeted regions and sequences with the m5C modifications on a transcriptome-wide scale15,16. The advantage of this protocol provides the comprehensive RNA m5C landscape at a single-base resolution since the antibody affinity enrichment-based approach relies upon the availability of high-quality antibodies and could achieve the single-fragment resolution of m5C methylation landscape17.
All RNA samples will be processed with two rounds of mRNA enrichment using oligo(dT) beads, three cycles of bisulfite reaction, and the sequencing library preparation. To monitor the RNA quality, each RNA sample will be examined by capillary gel electrophoresis before and after the procedures of mRNA purification and bisulfite reaction to assess fragment distribution. The purified libraries will be examined by their PCR amplicon qualities, DNA size distribution fragments by capillary gel electrophoresis, and their overall quantities examined by fluorescence dyes-based quantitative assays before sequencing. The system can also be used to analyze a broad spectrum of biological samples such as agricultural produce, isolated virions, cell lines, model organisms, and pathological specimens.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agilent 2100 Electrophoresis Bioanalyzer System</td>
			<td>Agilent, Santa Clara, CA</td>
			<td></td>
			<td>RNA quality detection</td>
		</tr>
		<tr>
			<td>AMpure XP beads</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
			<td>purify DNA</td>
		</tr>
		<tr>
			<td>Bioanalyzer DNA high sensitivity kit</td>
			<td>Agilent, Santa Clara, CA</td>
			<td>5067-4626</td>
			<td>DNA quality dection</td>
		</tr>
		<tr>
			<td>Bioanalyzer RNA 6000 Pico kit</td>
			<td>Agilent, Santa Clara, CA</td>
			<td>5067-1513</td>
			<td>RNA quality dection</td>
		</tr>
		<tr>
			<td>DiaMag02 - magnetic rack</td>
			<td>Diagenode, Denville, NJ</td>
			<td>B04000001</td>
			<td>assist library preparation</td>
		</tr>
		<tr>
			<td>DiaMag1.5 - magnetic rack</td>
			<td>Diagenode, Denville, NJ</td>
			<td>B04000003</td>
			<td>assist poly(A) RNA purificaion</td>
		</tr>
		<tr>
			<td>Dynabeads mRNA DIRECT purification kit</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>61011</td>
			<td>poly(A) RNA purificaion; Wash Buffer 1 and Wash Buffer 2</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>J.T.Baker</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>EZ RNA methylation kit</td>
			<td>Zymo, Irvine, CA</td>
			<td>R5002</td>
			<td>bisulfite treatment</td>
		</tr>
		<tr>
			<td>Firefly luciferase mRNA</td>
			<td>Promega, Madison, WI, USA</td>
			<td>L4561</td>
			<td>spike in control seqeunce&#160;</td>
		</tr>
		<tr>
			<td>KAPA Library Quantification Kits</td>
			<td>Roche, Switzerland</td>
			<td>KK4824</td>
			<td>library quantification</td>
		</tr>
		<tr>
			<td>Nanodrop spectrophotometer</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td></td>
			<td>Total RNA quantity detection</td>
		</tr>
		<tr>
			<td>NEBNext multiplex Oligos for illumina (index Primer set1)</td>
			<td>New England Biolabs, Ipswich, MA</td>
			<td>E7335S</td>
			<td>library preparation</td>
		</tr>
		<tr>
			<td>NEBNext Ultra <img alt="Equation 1" src="/files/ftp_upload/65352/65352eq01.jpg" style="margin: auto;" /> Directional RNA Library Prep Kit for Illumina</td>
			<td>New England Biolabs, Ipswich, MA</td>
			<td>E7760S</td>
			<td>library preparation</td>
		</tr>
		<tr>
			<td>Nuclease-free Water</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9932</td>
			<td></td>
		</tr>
		<tr>
			<td>P2 pipetman</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>4641010</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 2.0 fluorometer&#160;</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td></td>
			<td>RNA quantity detection</td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS Assay Kit</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>Q32854</td>
			<td>DNA quantity detection</td>
		</tr>
		<tr>
			<td>Qubit RNA HS Assay Kit</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>Q32852</td>
			<td>RNA quantity detection</td>
		</tr>
	</tbody>
</table>

enrichment of mrna and bisulfite-mrna library preparation for next-generation sequencing

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.

Author Spotlight: Decoding RNA Methylation&#039;s Role in Pancreatic Cancer - A Single-Base Resolution Study 

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

This research aims to study the role of RNA methylation, specifically 5-methylcytosine modification, in pancreatic cancer. The questions we want to answer are how upregulation or downregulation of certain RNA methyltransferase affects cancer cell phenotypes and also, through which RNA substrates. This study provides the comprehensive RNA m5C landscape at a single-base resolution with standardized equipment.From this study using bisulfite RNA seq, the results links the association of RNA m5C modification with poor differentiation of cancer cells. This should pave the way for future studies in discerning passenger versus disease driver effects of specific m5C in the RNA transcripts. With this technique, we can identify the set of differentially-methylated RNAs that may participate in tumor progression.Further, single molecule base experiments can provide a more detailed regulatory role of specific RNA m5C.

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...

Watch this Scientific Journal Video about Decoding RNA Methylation's Role in Pancreatic Cancer - A Single-Base Resolution Study at JoVE.com

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 ...

enrichment-mrna-bisulfite-mrna-library-preparation-for-next

Research

JoVE Journal

Biology

Poly(A) RNA Purification and Bisulfite Conversion for Next Generation Sequencing

To begin, prepare 10 to 20 micrograms of high-quality total RNA for the isolation of poly(A)enriched RNA. Transfer an aliquot of RNA into a nuclease free 1.5 milliliter microcentrifuge tube. Incubate the tube in a heat block at 70 degrees Celsius for five minutes, and then place it on ice for two minutes.After resuspending the oligo(dT)beads, transfer the required volume of bead suspension to a new 1.5 milliliter microcentrifuge tube and place it on the magnet for three minutes until the magnetic beads form a pellet. Remove the supernatant and add an equal volume of lysis buffer. Resuspend the beads.Place the tube back on the magnet for three minutes and remove the supernatant. For the first round of purification, add lysis buffer to the RNA sample tube and mix thoroughly. Then transfer the mixture to the oligo(dT)beads and resuspend fully pipetting at least 10 times.Allow the samples to incubate with continuous rotation at room temperature. After 15 minutes, place the sample on a magnet for five minutes or until the supernatant is clear. Discard the supernatant.Add 600 microliters of wash buffer one to the beads and carefully mix to resuspend. Next, add 300 microliters of wash buffer two to the beads and carefully mix to resuspend. Place the tube on the magnet for five minutes or until the supernatant is clear.Discard the supernatant. Next, add 30 microliters of cold 10 millimolar tris HCL to the sample tube and incubate at 70 degrees Celsius. After five minutes, quickly transfer the tube on the magnet.And when the suspension is clear, transfer the supernatant containing the eluted poly(A)enriched RNA into a new 1.5 milliliter microcentrifuge tube. For the second round of purification, add 120 microliters of lysis buffer to the eluted RNA sample and mix thoroughly. Wash the beads used in the first round of purification with an equal volume of lysis buffer.Pipette 10 times to resuspend the beads and then place the tube on the magnet for five minutes before discarding the supernatant. Transfer the RNA lysis buffer mixture to the washed beads and mix thoroughly by pipetting. After washing the beads as demonstrated previously, add 25 microliters of cold 10 millimolar tris HCL to the sample tube and incubate at 70 degrees Celsius.After five minutes, quickly transfer the tube to the magnet, and when the suspension is clear, transfer the supernatant containing eluted poly(a)enriched RNA to a new 1.5 milliliter microcentrifuge tube. Next, for bisulfite conversion, transfer 19 microliters of purified poly(A)enriched RNA sample to a 0.2 milliliter eight strip tube and add one microliter of spiking control at the predetermined one to 10, 000 quantity ratio. After adding 130 microliters of conversion reagent to the tubes and thoroughly mixing, place the tube in the PCR machine and start the PCR program.After PCR, place the column onto the collection tube and add 250 microliters of RNA binding buffer to the column. Then transfer 150 microliters of bisulfite treated sample to the column and pipette thoroughly. Add 400 microliters of 95 to 100%ethanol to the column, quickly close the cap and invert several times.After repeated centrifugation, washing, and neutralization with desulphonation buffer, transfer the column to a new 1.5 milliliter microcentrifuge tube. Add 20 to 30 microliters of nuclease-free water to the column and allow it to stand for one minute. Centrifuge at 10, 000 G for 30 seconds at 25 degrees Celsius and transfer 2.2 microliters of supernatant to eight strip tubes to assess RNA quantity and quality.Poly(A)RNA enrichment efficiency was assessed by capillary electrophoresis total RNA assay, resulting in decreased ribosomal RNA contamination after double purification in pancreatic cancer cell lines. RNA quantity before and after bisulfite treatment was assessed by capillary electrophoresis. After the bisulfite treatment, the RNA size distribution showed a 200 to 500 nucleotide peak because of fragmentation caused by the bisulfite reaction.Capillary electrophoresis of the amplicon showed successful library preparation with minimal primer remaining and no over amplification peak. After the sequencing reads aligned to the reference sequence, the average of 2, 440 of total reads were mapped to the spiking sequence, and the total analyzed C to T conversion rate reached an average of 99.81%

806 Views.  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.

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