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Single-Base RNA Modification Sequencing
Single-Nucleotide RNA m5C Modification Seq(BS-seq)
Single-Nucleotide RNA m5C Modification Seq(BS-seq) is an antibody-free, base-resolution method. Bisulfite conversion turns unmodified C into U (read as T) while m5C is protected and read as C, mapping and quantifying m5C at single-base resolution.
Arraystar Single-Nucleotide RNA m5C Modification Seq(BS-seq) is an end-to-end sample-to-data service for mRNA, from RNA sample QC and mRNA treatment through library construction, sequencing, and bioinformatics. Sites are annotated to the reference transcriptome with genomic coordinates, host gene, and functional region attribution.
Benefits
In mRNA, m5C influences stability and translation, so base-resolution mapping is informative. Bisulfite turns unmodified C into U (read as T) while m5C is protected and read as C, mapping each m5C site at base resolution.
Single-base resolution: pinpoints m5C sites in mRNA.
Quantitative stoichiometry: conversion-based readouts give accurate modification ratios.
Antibody-free and unbiased: avoids enrichment cross-reactivity and pull-down bias.
Functional annotation: Sites are annotated to the reference transcriptome with genomic coordinates, host gene, and functional region attribution.
Application-ready: supports translation control, RNA stability, coding-region function, and gene-regulation studies across the mRNA transcriptome.
| Service Name | RNA Class | Price |
|---|---|---|
| Single-Nucleotide RNA m5C Modification Seq(BS-seq) | mRNA | |
| Single-Nucleotide RNA m5C Modification Seq(BS-seq) | mRNA & lncRNA |
Background
In mRNA, the modification shapes transcript stability, translation, and gene regulation, so base-resolution mapping across coding and untranslated regions is what distinguishes it. Bisulfite conversion deaminates unmodified cytidine to uracil (read as thymine) while 5-methylcytidine is protected and read as cytosine, mapping each m5C site at single-base resolution. This antibody-free readout maps each m5C site at single-base resolution. The same workflow extends to long non-coding RNAs (lncRNAs), enabling researchers to profile this modification on lncRNA transcripts in parallel with mRNA in a single assay.
The method was validated for mRNA, sites are annotated to the reference transcriptome with genomic coordinates, host gene, and functional region attribution. (Dai Q, et al. He C. Nature Biotechnology 2024;42(10):1559-1570., DOI: 10.1038/s41587-023-02034-w).
Key concept: Single-Nucleotide RNA m5C Modification Seq(BS-seq) is a base-resolution method in which bisulfite conversion turns unmodified C into U (read as T) while m5C is protected and read as C, mapping and quantifying m5C at single-base resolution.
Figure 1. BS-seq m5C detection scheme. Bisulfite conversion turns unmodified C into U (read as T) while m5C is protected and read as C, mapping and quantifying m5C at single-base resolution.
Workflow
Five steps from RNA sample to single-base 5-methylcytidine (m⁵C) data:
Figure 2. Single-Nucleotide RNA m5C Modification Seq workflow.
Bioinformatics
The bioinformatics pipeline aligns reads to the reference transcriptome, detects C-to-T conversion signatures, and quantifies m5C stoichiometry at single-nucleotide resolution, with motif, distribution, differential methylation, enrichment, and genome-browser analyses.
Deliverables
- Raw m⁵C mRNA sequencing FASTQ data files, formatted for GEO/SRA public database submission.
- Sample and sequencing QC reports with sorted BAM alignment files and core mapping statistics.
- High-confidence single-base m⁵C site annotation tables (XLSX) with coordinates, genes, and absolute stoichiometry.
- Single-base m⁵C transcript feature distribution figures and consensus m⁵C motif logos (PDF/PNG format).
- Single-base resolution differential m⁵C methylation tables (XLSX) with fold change and significance metrics.
- Gene Ontology (GO) enrichment analysis reports for m⁵C-modified genes, provided in HTML/PDF/PNG formats.
- Genome browser-compatible track files (bigWig/bedGraph) and a full structured m⁵C analysis project report.
Research Applications
- Transcriptome-wide mapping of the modification across protein-coding and non-coding mRNA.
- Translational control: relates base-resolution modification to mRNA translation and stability.
- Coding-region function: localizes sites within CDS, UTR, and splice-adjacent regions.
- Disease-oriented studies: finds differentially modified mRNA sites as biomarkers.
References
[1] Frommer M, McDonald LE, Millar DS, et al. Paul CL. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. PNAS 1992;89(5):1827-1831. DOI: 10.1073/pnas.89.5.1827.
Sample Requirements
Storage Guidelines
Cells and Tissues: Preserve in TRIzol or an RNA stabilization solution; snap freeze in liquid nitrogen and store at –80 °C.
RNA: Resuspend in ethanol or RNase-free ultrapure water; store at –80 °C and avoid multiple freeze-thaw cycles.
Shipping Instructions
Place the sample in a 1.5 mL RNase-free microcentrifuge tube.
Seal the tube with parafilm or a cap lock to ensure sample integrity.
Ship the package on dry ice with adequate insulation to maintain the required temperature.
| Sample Type | Notes |
|---|---|
| Whole blood | Use EDTA tubes only, as heparin is not compatible with subsequent analytical procedures. |
| Cultured cells | Submission of cell pellets is preferred to ensure high-quality material for processing. |
| Tissue | Provide fresh or frozen specimens, ensuring that necrotic material is strictly avoided. |
| Total RNA | Maintain an OD 260/280 ratio ≥ 1.8 and RIN ≥ 7 with no visible degradation. Submit at least 10 µg total RNA. |
FAQ
Which mRNA features are annotated?
Sites are annotated to the reference transcriptome with genomic coordinates, host gene, and functional region attribution. This lets you interpret each site in the context of its host gene and mRNA functional region. The method supports both mRNA and tRNA workflows, so you can choose the readout that matches your study.
Can this method quantify the modification at single-base resolution?
Yes. Bisulfite conversion deaminates unmodified cytidine to uracil (read as thymine) while 5-methylcytidine is protected and read as cytosine, mapping each m5C site at single-base resolution. This provides single-base resolution and absolute stoichiometry for each site in mRNA.
How does this method differ from antibody-based mRNA profiling?
Antibody-based methods rely on enrichment and offer limited resolution with intrinsic motif bias. This method is antibody-free and delivers base-resolution, quantitative calling in mRNA, avoiding cross-reactivity and enrichment bias. This suits projects needing quantitative site-level mapping rather than region-level enrichment, and it pairs well with orthogonal validation.
How does this service support research on m5C modification?
By mapping m5C at base resolution, the service helps researchers examine its regulatory roles in RNA stability and expression, supporting high-impact studies.
Why is single-base m5C profiling valuable?
Precise m5C detection provides reliable site-level evidence that underpins strong conclusions and advances the field.
Does this service also profile lncRNA?
Yes. We provide two service options: mRNA modification, and mRNA plus long non-coding RNA (lncRNA) modification. Both transcript types can be analyzed together in a single experiment, so there is no need to split them into two projects, and the scope of your study opens up accordingly.
!This assay is provided for research applications only and is not intended for clinical diagnosis, treatment, or personal health evaluation.