Single-Nucleotide RNA m5C Modification Seq (BS-seq)
Bisulfite conversion reads m5C straight off the sequence: unmodified cytidine is converted and read as thymine, while m5C is protected and read as cytosine, which places every site and quantifies it at base resolution.
01What the service reports
Single-Nucleotide RNA m5C Modification Seq (BS-seq) is an antibody-free service that maps the positions of m5C in mRNA at base resolution and reports how large a share of the RNA at each position carries the modification.
The readout comes from a conversion rather than an enrichment. Unmodified cytidine is deaminated by bisulfite to uracil, which templates thymine in the sequencing reads; m5C resists the conversion and stays cytosine. The C-to-T pattern therefore marks modified positions one base at a time, and the fraction of reads that keep a C at that position gives the modification ratio.
The service is delivered as a complete sample-to-data workflow - RNA QC, bisulfite treatment, library construction, sequencing and bioinformatics - and every called site is annotated against the reference transcriptome with its coordinates, host gene and functional region.
02Benefits
- Base-resolution positions: m5C sites in mRNA are located individually instead of being confined to a converted region.
- Quantitative readout: conversion-based counting yields a modification ratio for each site rather than a qualitative call.
- Antibody-free: the readout does not depend on pull-down efficiency or on cross-reactivity of an antibody.
- Annotated output: each site arrives with genomic coordinates, its host gene and the functional region it falls in.
- Broadly usable results: the data serve studies of translation control, RNA stability, coding-region function and gene regulation 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 and lncRNA | |
| Single-Nucleotide RNA m5C Modification Seq (BS-seq) | rRNA |
03Background and detection principle
m5C in mRNA sits close to the machinery that decides how long a transcript lasts and how much protein it produces, and its influence plays out at individual positions rather than across whole transcripts. Enrichment-based profiling reports the latter: it indicates that a region carries the modification, but not which base does, and the signal follows the preferences of the reagent used.
Bisulfite sequencing answers at the level of the base. Deamination converts unmodified cytidine to uracil, read as thymine, while 5-methylcytidine is protected and read as cytosine, so the conversion pattern itself reveals the modified position and, from read counts, how much of the RNA is modified there. The same workflow extends to long non-coding RNA, so mRNA and lncRNA can be profiled side by side in one experiment.
Converting what is not modified is what makes the modification visible: the base that keeps its C is the base that carries m5C.Dai Q. et al., Nature Biotechnology 2024 · DOI 10.1038/s41587-023-02034-w
04Workflow
A BS-seq project moves through five stages:
- RNA QC to confirm that the sample is intact and clean enough for conversion.
- Bisulfite treatment, the step that separates modified from unmodified cytidine.
- Library construction from the converted RNA.
- Sequencing of the BS-seq library.
- Bioinformatics: C-to-T signature detection, per-site stoichiometry and annotation.
05Bioinformatics and deliverables
Reads are aligned to the reference transcriptome, C-to-T conversion signatures are detected, and the m5C stoichiometry of each site is quantified at single-nucleotide resolution. The analysis set adds motif characterisation, transcript feature distribution, differential methylation, enrichment analysis and genome-browser visualization.
Deliverables
- Raw m5C mRNA sequencing data files (FASTQ), formatted for GEO/SRA submission.
- Sample and sequencing QC reports, together with sorted BAM alignments and core mapping statistics.
- High-confidence single-base m5C site annotation tables (XLSX) with coordinates, host genes and stoichiometry.
- Transcript feature distribution figures and consensus m5C motif logos (PDF/PNG).
- Differential m5C methylation tables (XLSX) with fold change and significance metrics.
- GO enrichment reports for m5C-modified genes (HTML/PDF/PNG).
- Genome-browser-compatible tracks (bigWig/bedGraph) and a structured m5C analysis project report.
06Research applications
- Transcriptome-wide mapping: covers protein-coding transcripts and long non-coding RNAs in one study design.
- Translational control: links base-resolution m5C calls to mRNA translation and to transcript stability.
- Coding-region function: places sites within CDS, UTR and splice-adjacent regions so their position relative to the reading frame is explicit.
- Disease-oriented studies: differentially modified sites can be followed up as candidate biomarkers.
- Gene regulation: site-level ratios support comparisons of m5C between conditions, tissues or treatments without depending on an enrichment step.
07Sample requirements
Storage. Cells and tissues: preserve in TRIzol or an RNA stabilisation solution, snap freeze in liquid nitrogen and store at −80 °C. RNA: keep in ethanol or RNase-free ultrapure water at −80 °C and avoid repeated freeze–thaw cycles.
Shipping. Send the sample in a 1.5 mL RNase-free microcentrifuge tube, sealed with parafilm or a cap lock, on dry ice with adequate insulation.
| Sample type | Notes |
|---|---|
| Whole blood | EDTA tubes only; heparin is not compatible with the downstream procedure. |
| Cultured cells | Cell pellets are preferred. |
| Tissue | Fresh or frozen specimens; avoid necrotic material. |
| Total RNA | OD 260/280 ≥ 1.8 and RIN ≥ 7, with no visible degradation. Submit at least 10 µg total RNA. |
08FAQ
Which regions of a transcript are annotated for the called sites?
Sites are annotated against the reference transcriptome, so each one is reported with genomic coordinates, its host gene and the functional region it belongs to. That makes it possible to read a site in relation to its gene and to where it sits in the transcript.
Is the modification quantified, or only mapped?
Both. Unmodified cytidine is converted and read as T while m5C is read as C, so the conversion pattern locates each site and the read counts at that position give its modification ratio.
What is the difference from antibody-based mRNA profiling?
Antibody-based profiling works by enrichment, which limits resolution and introduces reagent bias. This service is antibody-free and produces base-resolution, quantitative calls, which suits projects that need site-level numbers and can be paired with orthogonal validation.
Can lncRNA be profiled as well?
Yes. Two scopes are offered: modification profiling on mRNA, and on mRNA plus long non-coding RNA. Both transcript types are handled inside one experiment instead of being split into two projects.
Which research questions benefit from a quantitative m5C map?
A quantitative m5C map supports work on transcript fate, local structure and condition-dependent remodelling on mRNA and long non-coding RNA. Sites that change in level, not only in detection, are the set that usually carries the biological claim.
09References
- Dai Q, Ye C, Irkliyenko I, et al. Ultrafast bisulfite sequencing detection of 5-methylcytosine in DNA and RNA. Nature Biotechnology 2024;42(10). DOI: 10.1038/s41587-023-02034-w
- Frommer M, McDonald LE, Millar DS, et al. 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