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Single-Base RNA Modification Sequencing
Single-Nucleotide RNA m3C Modification Seq(HAC-seq)
Single-Nucleotide RNA m3C Modification Seq(HAC-seq) is an antibody-free, base-resolution method. Hydrazine-aniline-mediated cleavage selectively acts on cytidine, and the resulting cleavage signature pinpoints N3-methylcytidine at single-base resolution.
Arraystar Single-Nucleotide RNA m3C Modification Seq(HAC-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, m3C can influence transcript stability and function, so base-resolution mapping is informative. Hydrazine-aniline cleavage acts on cytidine and the resulting signature pinpoints each m3C site at base resolution.
Single-base resolution: pinpoints m3C 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 m3C Modification Seq(HAC-seq) | mRNA | |
| Single-Nucleotide RNA m3C Modification Seq(HAC-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. Hydrazine-aniline-mediated cleavage selectively acts on cytidine so the resulting cleavage and reverse-transcription signature pinpoints N3-methylcytidine at single-base resolution. This antibody-free readout maps each m3C 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. (Cui J, Liu Q, Sendinc E, Shi Y, Gregory RI. Nucleic Acids Research 2021;49(5):e27., DOI: 10.1093/nar/gkaa1186).
Key concept: Single-Nucleotide RNA m3C Modification Seq(HAC-seq) is a base-resolution method in which hydrazine-aniline-mediated cleavage selectively acts on cytidine, and the resulting cleavage signature pinpoints N3-methylcytidine at single-base resolution.
Figure 1. HAC-seq m3C detection scheme. Hydrazine-aniline-mediated cleavage selectively acts on cytidine, and the resulting cleavage signature pinpoints N3-methylcytidine at single-base resolution.
Workflow
Five steps from RNA sample to single-base N3-methylcytidine (m³C) data:
Figure 2. Single-Nucleotide RNA m3C Modification Seq workflow.
Bioinformatics
The bioinformatics pipeline aligns reads to the reference transcriptome, recognizes HAC-seq cleavage signatures, and quantifies m3C stoichiometry at single-nucleotide resolution, with motif, differential, enrichment, and genome-browser analyses.
Deliverables
- Raw m³C 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 m³C site annotation tables (XLSX) with coordinates, genes, and absolute stoichiometry.
- Single-base m³C transcript feature distribution figures and consensus m³C motif logos (PDF/PNG format).
- Single-base resolution differential m³C methylation tables (XLSX) with fold change and significance metrics.
- Gene Ontology (GO) enrichment analysis reports for m³C-modified genes, provided in HTML/PDF/PNG formats.
- Genome browser-compatible track files (bigWig/bedGraph) and a full structured m³C 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] Cui J, Liu Q, Sendinc E, Shi Y, Gregory RI. Nucleotide resolution profiling of m3C RNA modification by HAC-seq. Nucleic Acids Research 2021;49(5):e27. DOI: 10.1093/nar/gkaa1186.
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 5–10 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. Hydrazine-aniline-mediated cleavage selectively acts on cytidine so the resulting cleavage and reverse-transcription signature pinpoints N3-methylcytidine at single-base resolution. This provides single-base resolution and absolute stoichiometry for each site in mRNA. The reaction is antibody-free and reproducible, so the calling is specific and the stoichiometry is reliable for downstream functional analyses.
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 m3C modification?
By mapping m3C at base resolution, the service helps researchers examine its influence on RNA stability and function, supporting high-impact mechanistic studies.
Why is high-resolution m3C profiling valuable?
Precise m3C mapping provides a trusted evidence base for translational research, advancing understanding of m3C roles.
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.