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 essentially follow the manufacturer&#39;s instructions with minor modifications indicated in the specific steps. See the Table of Materials for details related to all materials and instruments used in this protocol.
<ol>
	<li>Preparation
	<ol>
		<li>Warm up the reagent, washing buffer, oligo(dT) beads, and lysis buffer at room temperature for 30 min before conducting the following procedures.</li>
		<li>For the isolation of poly(A)-enriched RNA, prepare 10-20 &#181;g of an aliquot of high-quality total RNA as the starting material by transferring an adequate quantity of RNA into a nuclease-free 1.5 mL microcentrifuge tube. Incubate in the heat block at 70 &#176;C for 5 min, then keep on ice for 2 min. 
		NOTE: The quantities of total RNA as the starting material should be appropriately adjusted depending on the desired quantity of mRNA and the nature of the cells used. Empirically, the poly(A)-enriched RNA may account for 1-5% abundances in the total RNA. An aliquot of 10 &#181;g of high-quality total RNA, should provide high-quality poly(A)-enriched RNA in the 100-500 ng range for the downstream bisulfite conversion.</li>
		<li>Meanwhile, fully resuspend the oligo(dT) beads. Transfer the required amounts of beads suspension to a new 1.5 mL microcentrifuge tube, then place on the magnet for 3 min till the magnetic beads form a pellet. 
		NOTE: Use 50 &#181;L oligo(dT) beads for 10 &#181;g of total RNA input. The volume of beads in this step could be further optimized depending on the cells used.</li>
		<li>Remove the supernatant and resuspend the oligo(dT) beads with equal amounts (the volume of the beads suspension used in step 1.1.3.) of lysis buffer and place on the magnet for 3 min before removing the supernatant. 
		NOTE: Do not overdry the beads; the user should quickly proceed to step 1.2.1.</li>
	</ol>
	</li>
	<li>The first round of purification
	<ol>
		<li>Add lysis buffer to the RNA sample tube (volume ratio 4:1) and mix thoroughly before transferring to the oligo(dT) beads. 
		&#8203;NOTE: If the RNA sample volume is 20 &#181;L, then add 80 &#181;L of lysis buffer.</li>
		<li>Resuspend the RNA: lysis buffer mix and oligo(dT) beads fully by pipetting at least 10x.</li>
		<li>Allow the samples to incubate with continuous rotation for 15 min at room temperature.</li>
		<li>Place the sample on the magnet for 5 min or until the supernatant is clear; discard the supernatant.</li>
		<li>Add 600 &#181;L of Wash Buffer 1 to the beads and carefully mix to resuspend the beads.</li>
		<li>Place the tube on the magnet for 5 min or until the supernatant is clear, discard the supernatant, and repeat wash step 1.2.5 once.</li>
		<li>Wash the beads by adding 300 &#181;L of Wash Buffer 2, and gently resuspend the beads.</li>
		<li>Place the tube on the magnet for 5 min or until the supernatant is clear, discard the supernatant, and repeat wash step 1.2.7 once.</li>
		<li>Add 30 &#181;L of cold 10 mM Tris-HCl (Elution buffer) to the sample tube and incubate at 70 &#176;C for 5 min.</li>
		<li>Quickly transfer the tube onto the magnet, and when the suspension is clear after 20 s or more, transfer the supernatant containing eluted poly(A) enriched RNA to the new 1.5 mL microcentrifuge tube.</li>
	</ol>
	</li>
	<li>The second round of purification
	<ol>
		<li>Add 120 &#181;L (4x the volume of eluted sample) lysis buffer to the eluted RNA sample and mix thoroughly.</li>
		<li>Wash the beads used in the first round of purification with an equal amount of lysis buffer as step 1.1.4, pipet 10x to resuspend the beads, and then place them on the magnet for 5 min before discarding the supernatant.</li>
		<li>Transfer the RNA: lysis buffer mix to the washed beads and mix thoroughly by pipetting at least 10x.</li>
		<li>Incubate the sample with continuous rotation for 15 min at room temperature.</li>
		<li>Repeat steps 1.2.4 to 1.2.8 to remove salt and other RNA subtypes. 
		NOTE: The volume of washing buffer can be reduced to 300 &#181;L of Wash Buffer 1 and 150 &#181;L of Wash Buffer 2.</li>
		<li>Add 25 &#181;L (desired volume) of cold 10 mM Tris-HCl (Elution buffer) to the sample tube and incubate at 70 &#176;C for 5 min.</li>
		<li>Quickly transfer the tube onto the magnet, and when the suspension is clear after 20 s or more, transfer the supernatant containing eluted poly(A) enriched RNA to a new 1.5 mL microcentrifuge tube.</li>
		<li>Transfer 2.2 &#181;L to 8-strip tubes for quality assessment of RNA quantity and quality using RNA high-sensitivity assays.</li>
		<li>Store the sample at -20 &#176;C for short periods and -70 &#176;C for a longer period. 
		NOTE: This can be a pause point in the protocol.</li>
	</ol>
	</li>
</ol>
2. Bisulfite conversion
NOTE: The centrifugation steps were all performed at room temperature. The procedures are essentially performed accordingly to the manufacturer&#39;s instructions but with an additional step 2.2 for adding the spike-in control mRNA sequence(s) before the bisulfite reaction step 2.3.
<ol>
	<li>Transfer 19 &#181;L of purified poly(A) enriched RNA sample to a 0.2 mL 8-strip tube.</li>
	<li>Add 1.0 &#181;L of spike-in control, which is free from any modifications at the predetermined 1:10,000 quantity ratio (i.e., 0.1 ng of luciferase mRNA: 1 &#181;g of purified poly(A) enriched RNA) into the RNA sample of step 2.1. 
	NOTE: The spike-in control sequence can be Firefly luciferase RNA, Renilla luciferase, or in vitro transcribed RNA sequence with no methylated cytosines.</li>
	<li>Add 130 &#181;L of conversion regent to the tube and gently invert several times for thorough mixing.</li>
	<li>Place the tube in the PCR machine and perform three cycles of heating at 70 &#176;C for 10 min followed by incubating at 64 &#176;C for 45 min each and then a final step to cool down to 4 &#176;C.</li>
	<li>Place the column onto the collection tube and transfer 250 &#181;L of RNA binding buffer to the column.</li>
	<li>Transfer the sample from step 2.4&#160;to the column from step 2.5 and pipet thoroughly.</li>
	<li>Add 400 &#181;L of 95-100% of ethanol to the column, quickly close the cap, and invert several times.</li>
	<li>Centrifuge at 10,000 &#215; g for 30 s at 25 &#176;C and discard the flowthrough.</li>
	<li>Add 200 &#181;L of RNA wash buffer to the column.</li>
	<li>Centrifuge at 10,000 &#215; g for 30 s at 25 &#176;C and discard the flowthrough.</li>
	<li>Add 200 &#181;L of desulfonation buffer and incubate at room temperature for 30 min.</li>
	<li>Centrifuge at 10,000 &#215; g for 30 s at 25 &#176;C and discard the flowthrough.</li>
	<li>Add 400 &#181;L of RNA wash buffer and centrifuge at 10,000 &#215; g for 30 s at 25 &#176;C and discard the flowthrough. Repeat this step once.</li>
	<li>Centrifuge again at 10,000 &#215; g for 2 min at 25 &#176;C to completely remove the residual wash buffer. 
	NOTE: The other centrifugation step 2.14 for 2 min before elution is to completely remove the wash buffer. The wash step is performed twice to wash out the high-salt component from the bisulfite reagent and the desulfonation buffer on the column.</li>
	<li>Transfer the column to a new 1.5 mL microcentrifuge tube.</li>
	<li>Add 20-30 &#181;L of Nuclease-free H2O to the column and stand at room temperature for 1 min.</li>
	<li>Centrifuge at 10,000 &#215; g for 30 s at 25 &#176;C.</li>
	<li>Transfer 2.2 &#181;L to 8-strip tubes for the assessment of RNA quantity and quality.</li>
	<li>Store the bisulfite-treated RNA sample at -20 &#176;C for short periods and -70 &#176;C for a longer period. 
	&#8203;NOTE: This can be a pause point in the protocol.</li>
</ol>
3. Bisulfite-treated mRNA library preparation
NOTE: Follow the library preparation instruction protocol section 4 for use with purified mRNA or rRNA-depleted RNA. The first priming step should follow the FFPE RNA protocol since the bisulfite treatment fragmentizes the RNA. Perform every step in the laminar flow hood and add the reaction mixture on an ice-chilled cooling rack..
<ol>
	<li>Priming the input RNA
	<ol>
		<li>Transfer the desired amount of bisulfite-treated mRNA sample in a total maximum of 5 &#181;L or add Nuclease-free H2O to make a total of 5 &#181;L. 
		NOTE: A minimum of 10 ng of bisulfite-treated mRNA as input is recommended for this type of RNA library preparation and to generate the final molarity of the 4nM library product.</li>
		<li>Add 1 &#181;L of Random primer (lilac) to the sample and mix by pipetting.</li>
		<li>Place the sample tube in the PCR machine preset at 65 &#176;C and lid at 105 &#176;C for 5 min and hold at 4 &#176;C.</li>
	</ol>
	</li>
	<li>First-strand cDNA synthesis
	<ol>
		<li>Add 4 &#181;L of synthesis reaction buffer (lilac), 8 &#181;L of strand specificity reagent (brown), and 2 &#181;L of synthesis enzyme mix (brown) to the sample, which makes a total volume of 20 &#181;L. Mix thoroughly by pipetting at least 10x and quickly spin down.</li>
		<li>Place the sample tube in the PCR machine preset at 25 &#176;C for 10 min, 42 &#176;C for 50 min, 70 &#176;C for 15 min, and hold at 4 &#176;C. 
		NOTE: For inserts over 200 bases, a 42 &#176;C incubation period of 50 min is suggested; for insert size below 200 bases, a 42 &#176;C incubation period of 15 min is suggested.</li>
	</ol>
	</li>
	<li>Second-strand cDNA synthesis
	<ol>
		<li>Add 8 &#181;L of synthesis reaction buffer with dUTP mix (orange), 4 &#181;L of synthesis enzyme mix (orange), and 48 &#181;L of Nuclease-free H2O. Mix thoroughly by pipetting at least 10x and quickly spin down.</li>
		<li>Place the sample tube in the PCR machine preset at 16 &#176;C for 1 h with the lid set off or &#8804;40 &#176;C.</li>
	</ol>
	</li>
	<li>Purification of double-stranded cDNA with DNA purification beads
	<ol>
		<li>Prewarm the DNA purification beads 30 min before conducting the purification and vortex to resuspend the beads.</li>
		<li>Add 144 &#181;L of resuspended DNA purification beads to the cDNA sample from step 3.3.2&#160;and mix thoroughly by pipetting at least 10x.</li>
		<li>Incubate at room temperature for 5 min.</li>
		<li>Place the sample tube on a magnetic rack for 5 min until the solution is clear and then, remove the supernatant and keep the beads that contain DNA.</li>
		<li>Add 200 &#181;L of freshly made 80% ethanol to the tube with the beads while keeping the sample on the magnetic rack and incubate at room temperature for 30 s.</li>
		<li>Remove the supernatant and repeat step 3.4.5&#160;once, but this time carefully remove all the residual supernatant.</li>
		<li>Air dry the beads for 1-5 min while keeping the sample on the magnet. 
		NOTE: Do not overdry the beads; the color should be dark brown.</li>
		<li>Remove the sample from the magnetic rack and elute the DNA by adding 53 &#181;L of 0.1x TE (10 mM Tris-HCl and 1 mM EDTA, pH 8.0) buffer to the beads. Mix thoroughly by pipetting at least 10x and incubate at room temperature for 2 min.</li>
		<li>Place the tube onto the magnetic rack for 5 min or until the solution is clear.</li>
		<li>Transfer 50 &#181;L of supernatant containing double-stranded cDNA to a new 8-strip tube. 
		NOTE: The protocol can be stopped at this point and the sample can be stored at -30 &#176;C.</li>
	</ol>
	</li>
	<li>End preparation of cDNA library
	<ol>
		<li>Add 7 &#181;L of end-polishing Reaction Buffer (green) and 3 &#181;L of the end-polishing Enzyme Mix (green) to the eluted double-stranded cDNA sample.</li>
		<li>Mix the mixture by pipetting 10x with a 50 &#181;L volume setting without creating bubbles, then quickly spin down the sample.</li>
		<li>Place the sample in a PCR machine and incubate at 20 &#176;C for 30 min, 65 &#176;C for 30 min, and hold at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Adaptor ligation
	<ol>
		<li>Dilute the adaptor (red) according to the manufacturer&#39;s instructions.</li>
		<li>Add 2.5 &#181;L of the diluted adaptor, 1 &#181;L of ligation enhancer (red), and 30 &#181;L of Ligation Master Mix (red) to the sample.</li>
		<li>Mix the mixture by pipetting 10x with an 80 &#181;L volume setting without creating bubbles, then quickly spin down the sample.</li>
		<li>Place the sample in a PCR machine and incubate at 20 &#176;C for 15 min.</li>
		<li>Add 3 &#181;L of the mixture of uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII (blue) to the sample and pipet thoroughly.</li>
		<li>Place back the sample in the PCR machine and incubate at 37 &#176;C for 15 min with the lid set to 75 &#176;C.</li>
	</ol>
	</li>
	<li>Purification of ligation reaction treated cDNAs 
	NOTE: The size selection with SPRIselect Beads, or optionally with AMpure XP beads can both be used for this purpose18.
	<ol>
		<li>Prewarm the beads 30 min before conducting the purification and vortex to resuspend them.</li>
		<li>Transfer 25 &#181;L of resuspended beads to the cDNA sample from step 3.6.6 and mix thoroughly by pipetting at least 10x.</li>
		<li>Incubate at room temperature for 5 min.</li>
		<li>Place the sample tube on a magnetic rack for 5 min until the solution is clear and then transfer the supernatant to a new 8-strip tube and discard the beads.</li>
		<li>Add 10 &#181;L of resuspended beads to the supernatant and mix thoroughly by pipetting at least 10x.</li>
		<li>Place the sample tube on a magnetic rack for 5 min until the solution is clear and then discard the supernatant and do not disturb the beads.</li>
		<li>Add 200 &#181;L of freshly made 80% ethanol to the tube with the beads while keeping the sample on the magnetic rack and incubate at room temperature for 30 s.</li>
		<li>Remove the supernatant and repeat step 3.7.7 once, but this time carefully remove all the residual supernatant.</li>
		<li>Air-dry the beads for 1-5 min while keeping the sample on the magnet. 
		&#8203;NOTE: Do not overdry the beads; the color should be dark brown.</li>
		<li>Remove the sample from the magnetic rack and elute the DNA by adding 17 &#181;L of 0.1x TE buffer to the beads. Mix thoroughly by pipetting at least 10x and incubate at room temperature for 2 min.</li>
		<li>Place the tube onto the magnetic rack for 5 min until the solution is clear.</li>
		<li>Transfer 15 &#181;L of supernatant, which contains target end-prepared dsDNA to a new 8-strip tube.</li>
	</ol>
	</li>
	<li>PCR enrichment of adaptor-ligated DNA
	<ol>
		<li>Add 25 &#181;L of master mix (blue), 5 &#181;L of 5&#39; adaptor primer, and 5 &#181;L of the 3&#39; adaptor primer to the adaptor-ligated DNA sample. Mix thoroughly by pipetting at least 10x.</li>
		<li>Place the sample in the PCR machine and perform PCR amplification using the following steps: (1) 98 &#176;C for 30 s, (2) 98 &#176;C for 10 s, (3) 65 &#176;C for 75 s, (4) 65 &#176;C for 5 min, and (5) hold at 4 &#176;C; in which the steps (2) and (3) should repeat for an N+2 cycle number where N equals to the RNA input quantity in the instruction manual. This means 2 additional cycles are needed due to the double-side size selection of samples performed in step 3.7. 
		NOTE: For instance, 8 ng of the purified mRNA input would be recommended with 9-10 cycles of PCR; but with double-side, size-selected, ligated cDNA in step 3.7, the recommended PCR cycle would be 11-12 cycles.</li>
	</ol>
	</li>
	<li>Purification of PCR-amplified libraries
	<ol>
		<li>Prewarm the DNA purification beads for 30 min before conducting the purification and vortex to resuspend the beads.</li>
		<li>Transfer 45 &#181;L (0.9x) of resuspended beads to the PCR product from step 3.8.2 and mix thoroughly by pipetting at least 10x.</li>
		<li>Incubate at room temperature for 5 min.</li>
		<li>Place the sample tube on a magnetic rack for 5 min until the solution is clear and then remove the supernatant and keep the beads that contain DNA.</li>
		<li>Add 200 &#181;L of freshly made 80% ethanol to the tube with the beads, leave the sample on the magnetic rack and allow it to stand at room temperature for 30 s.</li>
		<li>Remove the supernatant and repeat step 3.9.5 once, but this time carefully remove all the residual supernatant.</li>
		<li>Air-dry the beads for 1-5 min while keeping the sample on the magnet. 
		NOTE: Do not overdry the beads; the color should be dark brown.</li>
		<li>Take the sample off the magnetic rack and add 22 &#181;L of 0.1x TE buffer to the beads to elute the DNA. Mix thoroughly by pipetting at least 10x and incubate at room temperature for 2 min.</li>
		<li>Place the tube onto the magnetic rack and allow it to stand for 5 min or until the solution is clear at room temperature.</li>
		<li>Transfer 20 &#181;L of supernatant to a new 8-strip tube.</li>
		<li>Transfer 2.2 &#181;L to 8-strip tubes for quality assessment of library quantity and quality. 
		NOTE: The quantity of the library can also be detected by qPCR (see the Table of Materials).</li>
		<li>Store the bsRNA-seq library sample at -20 &#176;C.</li>
	</ol>
	</li>
</ol>

Creating Robust Bisulfite-mRNA Libraries for Transcriptome-Wide Mapping

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(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 conducting the poly(A) RNA purification step. Another crucial part of the procedure is the number of PCR cycles in library preparation. The decision of cycle number depends on the quantity of bisulfite-treated mRNA used for library preparation and whether the use of one size selection or double size selection in the step before PCR. The suitable cycle numbers for the sample of interest would thus require a trial run and assess the PCR product size distribution by capillary electrophoresis to determine the optimal cycle number for the same cell line or tissue.
In this protocol, two rounds of oligo(dT) beads purification were used to enrich and purify poly(A) RNA and eliminated a majority of ribosomal RNAs and other RNAs. There are other methods to remove ribosomal RNA and keep other RNA types in the sample, such as oligo-based depletion of rRNA26. Then, the 5-methylcytosine modification present in other RNA types can also be taken into analysis and extend the understanding of 5-methylcytosine features in mRNA and other RNAs.
Bisulfite reaction is known for not being able to distinguish m5C from other modifications, including the 5-hydroxymethylcytosine (hm5C), 3-methylcytidine (m3C), and N4-methylcytidine (m4C)27. However, the experimental design including m5C RNA methyltransferase knockdown or knockout should provide informative evidence of high-confidence regulated m5C sites. Additionally, validation using different methods such as antibody-based enrichment followed by sequencing or PCR or LC-MS/MS-based methods could essentially authenticate the candidate m5C sites16. The emerging technique of direct Nanopore RNA sequencing also provides potential identification of RNA modification via the computational analysis of the current signal or base-called &#39;error&#39; features28,29,30.
A recent study by Johnson et al. showed that adding the fragmentation step in mitochondria RNA samples after three rounds of bisulfite conversion indeed showed a higher conversion rate and increased&#160;the yield of cDNA library product31. This paper specifically analyzed the conversion rate and reads quality mapped to the mitochondria genome, not the mRNA transcriptome. Hence, the use of the fragmentation step has been updated in Table 1 to highlight whether the published reports included an RNA fragmentation step so that the viewers can easily replicate the protocol. Another study by Zhang et al. demonstrated that utilizing in vitro transcribed modification-free RNA library as a negative control is efficient to eliminate the false-positive signal from bisulfite treatment32. Recent studies have compared different protocols used for library construction33. The user can further choose to conduct bsRNA-seq&#160;pipeline with or without an additional fragmentation step and in vitro transcribed modification-free RNA library to customize suitable workflow.
The transcriptome-wide identification of m5C by different principles could strengthen the findings of the modification landscapes and provide more understanding of the regulatory mechanisms of RNA modifications in healthy or disease models.

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><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 detection</td></tr><tr><td>Bioanalyzer RNA 6000 Pico kit</td><td>Agilent, Santa Clara, CA</td><td>5067-1513</td><td>RNA quality detection</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&amp;nbsp;</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 &lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/65352/65352eq01.jpg&quot; style=&quot;margin: auto;&quot;/&gt; 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&amp;nbsp;</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.

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 purification efficiency of poly(A) RNA was assessed by capillary electrophoresis total RNA assay that can automatically calculate the rRNA contamination percentage (Figure 2). From the RNA fragment peak pattern and rRNA contamination percentage, RNA samples with twice purification indeed showed decreased contamination of ribosomal RNA from 6.5% to 2.2% and 2.6% to 1.1% in AsPC-1 and BxPC-3, respectively. With human pancreatic cancer cell lines, two rounds of beads purification could reach an average of 2.8 &#177; 1% poly(A)-enriched RNA abundances from the total RNA samples. Therefore, the poly(A) RNA enrichment by oligo(dT) beads with double purification steps minimized ribosomal RNA and represents a feasible mRNA sample enrichment for the downstream experiments.
The bisulfite conversion protocols had been reported in several studies on RNA 5-methylcytosine modification (Table 1). The bisulfite reaction could be performed with a user-reconstituted reaction mixture consisting of 40% sodium bisulfite and 600 mM hydroquinone in the aqueous solution (pH 5.1) at 75 &#176;C for 4 hours10,20,21. Alternatively, a more stringent bisulfite treatment of RNA sample may also be performed with a commercial kit; which in a number of studies from others and ours6,22,23,24 extended the bisulfite reaction time to three incubation cycles. Since the three-cycle incubation step efficiently converts unmethylated cytosine in the in vitro transcribed and structurally more-folded Escherichia coli 16S rRNA segment8, the three-cycle incubation step was also applied in this protocol.
With twice poly(A) enrichment and the three-cycle bisulfite conversion, RNA quality before and after the reaction was assessed by capillary electrophoresis. The RNA size distribution after the bisulfite treatment showed a peak of ~200-500 nucleotides because of fragmentation caused by the bisulfite reaction (Figure 3). Additionally, the fragmented bsRNA is ideal for library preparation without the need to conduct another fragmentation step. A total of 8-10 ng of bsRNA as input was used to construct the library according to this protocol by using 9 to 11 cycles in the final PCR amplification of adapter-ligated DNA. The capillary electrophoresis of the amplicon showed quite a successful library preparation, with only a small portion of primer remaining and no peak of over-amplification (Figure 4). To check the conversion efficiency in each sample library, the unmethylated firefly luciferase RNA as a spike-in control sequence was added to the sample before conducting the bisulfite treatment. After the sequencing reads aligned to the reference sequence using meRanTK tool kits25, the average of 2,440 (0.006%) of total reads were mapped to the spike-in sequence, and the total analyzed C-to-T conversion rate reached an average of 99.81%; the status can be viewed in Integrated Genomics Viewer (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65352/65352fig01.jpg" /> 
Figure 1: Workflow diagram of bisulfite mRNA sequencing. The pipeline constitutes of three main protocols, including mRNA enrichment, bisulfite conversion, and library preparation. Abbreviation: bsRNA = bisulfite mRNA; QC. = quality control. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65352/65352fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65352/65352fig02.jpg" /> 
Figure 2: Quality control results of poly(A)-enriched RNA samples with capillary electrophoresis. (A) The representative capillary electrophoresis profile of the total RNA from BxPC-3 cells. (B) The representative capillary electrophoresis profiles of samples processed with oligo(dT) purification from AsPC-1 and BxPC-3 cells. mRNA E1, mRNA elution from one-time purification; mRNA E2, mRNA elution from two-times purification. The rRNA contamination estimates were calculated by the electrophoresis operation system that controls the total RNA analysis assay. (C) The capillary electrophoresis profile of the Ladder markers was obtained in the same run with samples presented in panel B. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65352/65352fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65352/65352fig03.jpg" /> 
Figure 3: Quality assessment of RNA samples by capillary electrophoresis. The representative capillary electrophoresis profiles of (A) the total RNA, (B) the poly(A)-enriched mRNA from two times purification, and (C) the bisulfite-treated mRNA from the BxPC-3 cells. Abbreviations: B_empty = BxPC-3 cells transduced with empty vector; B_shScr = BxPC-3 cells transduced with the scramble sequences. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65352/65352fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65352/65352fig04.jpg" /> 
Figure 4: Quality assessment of bsRNA-seq&#160;libraries. (A-C) Three sets of representative capillary electrophoresis profiles of constructed bsRNA-seq&#160;libraries amplified by PCR at different PCR cycle numbers of 9 and 11 cycles using bsRNA from a set of BxPC-3 cells. The estimated input quantity of the bisulfite-treated RNA was determined by a high-sensitivity RNA quantification assay. A total of 8, 8.4, and 9.6 ng bsRNA were used in the (A) BxPC-3 mock, (B) BxPC-3 empty, and (C) BxPC-3 shN2UN2 samples, respectively. The ladder marker peaks at 35 bp and the 10,380 bp represent the internal standards of the lower- and upper-boundary in the electrophoresis profile of each sample. A prominent peak of around 100 bp and an obscure peak of around 127 bp, respectively, indicate the PCR primers (&#60;100 bp) and the adaptor-dimer (~127 bp), which remained in the eluate&#160;after PCR cleanup. Abbreviations: BxPC-3 mock = untransduced BxPC-3 cells; BxPC-3 empty = BxPC-3 cells transduced with the empty vector control; BxPC-3 shN2UN2 = BxPC-3 cells transduced with the shNSUN2 constructs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65352/65352fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65352/65352fig05.jpg" /> 
Figure 5: The sequence alignment profiles of each bsRNA-seq&#160;library mapped to the spike-in-control reference sequence. The representative alignment profiles of the BxPC-3 mock, empty, scramble, and shNSUN2 bsRNA-seq&#160;libraries to the firefly luciferase spike-in control reference sequence (indicated as the &#34;Z&#34; gene); 2 representative regions were shown. Grey bars indicate all reads that present matched consensus nucleotides at the indicated base position to the reference sequence. Red bars highlight the thymidine (T) identity at the base position of the reads; blue bars indicate those reads having the cytosine (C) identity at the position. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65352/65352fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Comparison of bisulfite reaction protocols and the conversion rate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65352/65352_JoVE_Table1 (1).xlsx" target="_blank">Please click here to download this Table.</a>

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

Author Spotlight: Decoding RNA Methylation's Role in Pancreatic Cancer - A Single-Base Resolution Study 

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.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Biology

1.0K Views.  National Cheng Kung University. Dit onderzoek heeft tot doel de rol van RNA-methylering, in het bijzonder 5-methylcytosinemodificatie, bij alvleesklierkanker te bestuderen. De vragen die we willen beantwoorden zijn hoe op- of neerwaartse regulatie van bepaalde RNA-methyltransferase de fenotypes van kankercellen beïnvloedt en ook via welke RNA-substraten. Deze studie biedt het uitgebreide RNA m5C-landschap met een resolutie met één basis met gestandaardiseerde apparatuur.Uit deze studie met bisulfiet RNA seq blijkt...

Video: Auteur in de schijnwerpers: de rol van RNA-methylering bij alvleesklierkanker decoderen - een onderzoek met één basisresolutie 

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of cancer development. By using non-invasive or pre-malignant lesions from patients who later develop invasive disease, gene expression analyses may help identify early molecular alterations that predispose to cancer risk. It has been well described that nucleic acids recovered from FFPE tissues have undergone severe physical damage and chemical modifications, which make their analysis difficult and generally requires adapted assays. MicroRNAs (miRNAs), however, which represent a small class of RNA molecules spanning only up to ~18&#8211;24 nucleotides, have been shown to withstand long-term storage and have been successfully analyzed in FFPE samples. Here we present a 3&#39; barcoded complementary DNA (cDNA) library preparation protocol specifically optimized for the analysis of small RNAs extracted from archived tissues, which was recently demonstrated to be robust and highly reproducible when using archived clinical specimens stored for up to 35 years. This library preparation is well adapted to the multiplex analysis of compromised/degraded material where RNA samples (up to 18) are ligated with individual 3&#39; barcoded adapters and then pooled together for subsequent enzymatic and biochemical preparations prior to analysis. All purifications are performed by polyacrylamide gel electrophoresis (PAGE), which allows size-specific selections and enrichments of barcoded small RNA species. This cDNA library preparation is well adapted to minute RNA inputs, as a pilot polymerase chain reaction (PCR) allows determination of a specific amplification cycle to produce optimal amounts of material for next-generation sequencing (NGS). This approach was optimized for the use of degraded FFPE RNA from specimens archived for up to 35 years and provides highly reproducible NGS data.

Formalin-fixed paraffin-embedded specimens represent a valuable source of molecular biomarkers of human diseases. Here we present a laboratory-based cDNA library preparation protocol, initially designed with fresh frozen RNA, and optimized for the analysis of archived microRNAs from tissues stored up to 35 years.

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of ...

Genetics

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as plant microRNAs (miRNAs) carry a 2&#39;-O-methyl (2&#39;-OMe) modification at their 3&#39;&#160;terminal nucleotide. This modification adds another difficulty as it inhibits 3&#39;&#160;adapter ligation. We previously demonstrated that modified versions of the &#39;TruSeq (TS)&#39;&#160;protocol have less bias and an improved detection of 2&#39;-OMe RNAs. Here we describe in detail protocol &#39;TS5&#39;, which showed the best overall performance. TS5 can be followed either using homemade reagents or reagents from the TS kit, with equal performance.

We present a detailed small RNA library reparation protocol with less bias than standard methods and an increased sensitivity for 2&#39;-O-methyl RNAs. This protocol can be followed using homemade reagents to save cost or using kits for convenience.

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as ...

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a complementary DNA (cDNA) intermediate. However, such approaches are rarely applied as a de novo RNA sequencing method, but used mainly as a tool that can assist in quality assurance for confirming known sequences of purified single-stranded RNA samples. Recently, we developed&#160;a direct RNA sequencing method by integrating a 2-dimensional mass-retention time hydrophobic end-labeling strategy into MS-based sequencing (2D-HELS MS Seq). This method is capable of accurately sequencing single RNA sequences as well as mixtures containing up to 12 distinct RNA sequences. In addition to the four canonical ribonucleotides (A, C, G, and U), the method has the capacity to sequence RNA oligonucleotides containing modified nucleotides. This is possible because the modified nucleobase either has an intrinsically unique mass that can help in its identification and its location in the RNA sequence, or can be converted into a product with a unique mass. In this study, we have used RNA, incorporating two representative modified nucleotides&#160;(pseudouridine (&#936;) and 5-methylcytosine (m5C)), to illustrate the application of the method for the de novo sequencing of a single RNA oligonucleotide&#160;as well as a mixture of RNA oligonucleotides, each with a different sequence and/or modified nucleotides. The procedures and protocols described here to sequence these model RNAs will be applicable to other short RNA samples (&#60;35 nt) when using a standard high-resolution LC-MS system, and can also be used for&#160;sequence verification of modified therapeutic RNA oligonucleotides.&#160;In the future, with the development of more robust algorithms and with better instruments, this method could allow sequencing of more complex biological samples.&#160;

Here, we describe a detailed protocol for an LC-MS-based sequencing method that can be used as a direct method to sequence short RNA (&#60;35 nt per run) without a cDNA intermediate, and as a general method to sequence different nucleotide modifications in a single study at single-base precision.

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a ...

Analyzing tRNA Dynamics Using AQRNA-seq

AQRNA-seq for Quantifying Small RNAs

AQRNA-seq provides a direct linear relationship between sequencing read counts and small RNA copy numbers in a biological sample, thus enabling accurate quantification of the pool of small RNAs. The AQRNA-seq library preparation procedure described here involves the use of custom-designed sequencing linkers and a step for reducing methylation RNA modifications that block reverse transcription processivity, which results in an increased yield of full-length cDNAs. In addition, a detailed implementation of the accompanying bioinformatics pipeline is presented. This demonstration of AQRNA-seq was conducted through a quantitative analysis of the 45 tRNAs in Mycobacterium bovis BCG harvested on 5 selected days across a 20-day time course of nutrient deprivation and 6 days of resuscitation. Ongoing efforts to improve the efficiency and rigor of AQRNA-seq will also be discussed here. This includes exploring methods to obviate gel purification for mitigating primer dimer issues after PCR amplification and to increase the proportion of full-length reads to enable more accurate read mapping. Future enhancements to AQRNA-seq will be focused on facilitating automation and high-throughput implementation of this technology for quantifying all small RNA species in cell and tissue samples from diverse organisms.

Author Spotlight: AQRNA-seq Role in Mapping Small RNAs and Unraveling Protein Translation Mechanisms

Absolute quantification RNA sequencing (AQRNA-seq) is a technology developed to quantify the landscape of all small RNAs in biological mixtures. Here, both the library preparation and data processing steps of AQRNA-seq are demonstrated, quantifying changes in the transfer RNA (tRNA) pool in Mycobacterium bovis BCG during starvation-induced dormancy.

AQRNA-seq provides a direct linear relationship between sequencing read counts and small RNA copy numbers in a biological sample, thus enabling accurate ...

RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of this method, in comparison to other approaches, is its ability to monitor translation by precisely mapping the position and number of ribosomes on a mRNA transcript.
In this article, we describe the consecutive stages of sample collection and preparation for RIBO-seq method in bacteria, highlighting the details relevant to the planning and execution of the experiment.
Since the RIBO-seq relies on intact ribosomes and related mRNAs, the key step is rapid inhibition of translation and adequate disintegration of cells. Thus, we suggest filtration and flash-freezing in liquid nitrogen for cell harvesting with an optional pretreatment with chloramphenicol to arrest translation in bacteria. For the disintegration, we propose grinding frozen cells with mortar and pestle in the presence of aluminum oxide to mechanically disrupt the cell wall. In this protocol, sucrose cushion or a sucrose gradient ultracentrifugation for monosome purification is not required. Instead, mRNA separation using polyacrylamide gel electrophoresis (PAGE) followed by the ribosomal footprint excision (28-30 nt band) is applied and provides satisfactory results. This largely simplifies the method as well as reduces the time and equipment requirements for the procedure. For library preparation, we recommend using the commercially available small RNA kit for Illumina sequencing from New England Biolabs, following manufacturer&#39;s guidelines with some degree of optimization.
The resulting cDNA libraries present appropriate quantity and quality required for next generation sequencing (NGS). Sequencing of the libraries prepared according to the described protocol results in 2 to 10 mln uniquely mapped reads per sample providing sufficient data for comprehensive bioinformatic analysis. The protocol we present is quick and relatively easy and can be performed with standard laboratory equipment.

Here we describe the stages of sample collection and preparation for RIBO-seq in bacteria. Sequencing of the libraries prepared according to these guidelines results in sufficient data for comprehensive bioinformatic analysis. The protocol we present is simple, uses standard laboratory equipment and takes seven days from lysis to obtaining libraries.

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of ...

Biochemistry

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

Developmental Biology

Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other genome-wide analyses. mRNA-Seq even opens the gate for gene expression analysis of non-sequenced genomes. mRNA-Seq offers high sensitivity, a large dynamic range and allows measurement of transcript copy numbers in a sample. Illumina&#x2019;s genome analyzer performs sequencing of a large number (&gt; 107) of relatively short sequence reads (&lt; 150 bp).The &quot;paired end&quot; approach, wherein a single long read is sequenced at both its ends, allows for tracking alternate splice junctions, insertions and deletions, and is useful for de novo transcriptome assembly.
One of the major challenges faced by researchers is a limited amount of starting material. For example, in experiments where cells are harvested by laser micro-dissection, available starting total RNA may measure in nanograms. Preparation of mRNA-Seq libraries from such samples have been described1, 2 but involves significant PCR amplification that may introduce bias. Other RNA-Seq library construction procedures with minimal PCR amplification have been published3, 4 but require microgram amounts of starting total RNA.
Here we describe a protocol for the Illumina Genome Analyzer II platform for mRNA-Seq sequencing for library preparation that avoids significant PCR amplification and requires only 10 nanograms of total RNA. While this protocol has been described previously and validated for single-end sequencing5, where it was shown to produce directional libraries without introducing significant amplification bias, here we validate it further for use as a paired end protocol. We selectively amplify polyadenylated messenger RNAs from starting total RNA using the T7 based Eberwine linear amplification method, coined &quot;T7LA&quot; (T7 linear amplification). The amplified poly-A mRNAs are fragmented, reverse transcribed and adapter ligated to produce the final sequencing library. For both single read and paired end runs, sequences are mapped to the human transcriptome6 and normalized so that data from multiple runs can be compared. We report the gene expression measurement in units of transcripts per million (TPM), which is a superior measure to RPKM when comparing samples7.

Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other ...

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

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

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