SINGLE-BASE RNA MODIFICATION SEQUENCING

Single-Nucleotide RNA ac⁴C Modification Seq (RedaC:T-seq)

An antibody-free route to ac⁴C: a reductive RedaC:T conversion acts on acetylated cytidine and writes it into the read as a defined signature, leaving unmodified cytidine distinguishable, so each site is placed at an individual nucleotide.

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01What the service reports

Single-Nucleotide RNA ac⁴C Modification Seq (RedaC:T-seq) is a base-resolution method in which a reductive RedaC:T conversion selectively acts on acetylated cytidine and records it as a readable signature while leaving unmodified cytidine distinguishable.

The readout is per site: a table of ac⁴C positions across protein-coding transcripts, each with genomic coordinates, host gene, functional region and a modification ratio. Because the signal comes from a conversion rather than from an enrichment step, there is no antibody in the measurement.

Arraystar runs RedaC:T-seq as a sample-to-data service: RNA QC, the reductive conversion, library construction, sequencing and bioinformatics. Two transcript scopes are offered, so ac⁴C can be profiled on mRNA alone or on mRNA together with long non-coding RNA in the same experiment.

02Benefits

Single-base resolution: each ac⁴C site is placed at an individual cytidine rather than inside an enriched window.
Site-level stoichiometry: the conversion-based readout reports a modification ratio for every called site.
Antibody-free: no enrichment step, so antibody cross-reactivity and pull-down bias are not sources of variation; the measurement does not rely on those steps.
Functional annotation: sites are mapped to the reference transcriptome with genomic coordinates, host gene and functional region attribution.
Built for mechanism work: the site table supports studies of translation control, transcript stability and coding-region function.

Service Name	RNA Class	Price
Single-Nucleotide RNA ac4C Modification Seq (RedaC:T-seq)	mRNA
Single-Nucleotide RNA ac4C Modification Seq (RedaC:T-seq)	mRNA and lncRNA
Single-Nucleotide RNA ac4C Modification Seq (RedaC:T-seq)	rRNA

03Background and detection principle

On mRNA, ac⁴C has been linked to transcript stability, translation and gene regulation. Much of that work turns on where the site sits — coding sequence, untranslated region or near a splice junction — which is exactly what an enrichment-based profile cannot resolve.

RedaC:T-seq answers that with chemistry instead of an antibody. A reductive conversion acts selectively on acetylated cytidine and records it as a readable signature, while unmodified cytidine is distinguished in the same read. The result is a quantitative, single-base map across protein-coding transcripts, and the same workflow can be extended to lncRNA in the same experiment rather than as a separate project.

RedaC:T-seq ac4C detection scheme — **Figure 1.** RedaC:T-seq detection scheme. The reductive conversion acts on acetylated cytidine and records it as a readable signature, so ac⁴C is mapped and quantified at single-base resolution.

The site, not the window, is what the experiment reports — which is what makes ac⁴C readable in the context of its host transcript.Arango D. et al., Molecular Cell 2022;82(15):2797–2814 · DOI 10.1016/j.molcel.2022.05.016

04Workflow

Five steps take a sample from RNA to a single-base ac⁴C table:

RNA sample QC, covering integrity and purity.
Reductive RedaC:T conversion of the RNA.
Library construction from the converted material.
Sequencing of the RedaC:T-seq library.
Bioinformatics: conversion signature calling, site annotation and stoichiometry estimation.

**Figure 2.** RedaC:T-seq workflow, from RNA sample to single-base ac⁴C data.

05Bioinformatics and deliverables

Reads are aligned to the reference transcriptome, RedaC:T conversion signatures are recognized, and ac⁴C stoichiometry is estimated per site. The analysis set covers motif characterisation, differential modification, functional enrichment and genome-browser visualization.

Deliverables

Raw ac⁴C mRNA sequencing data files (FASTQ), formatted for GEO/SRA submission.
Sample and sequencing QC reports, sorted BAM alignment files and core mapping statistics.
High-confidence single-base ac⁴C site tables (XLSX) with coordinates, host gene and stoichiometry.
Single-base transcript feature distribution figures and consensus ac⁴C motif logos (PDF/PNG).
Single-base differential ac⁴C tables (XLSX) with fold change and significance.
GO enrichment reports for ac⁴C-modified genes (HTML/PDF/PNG).
Genome-browser track files (bigWig/bedGraph) and a structured project report.

06Research applications

Transcriptome-wide mapping: ac⁴C sites resolved across protein-coding transcripts and, when the lncRNA scope is selected, across lncRNA as well.
Translational control: relates a resolved site to mRNA translation and stability.
Coding-region function: localises sites within CDS, UTR and splice-adjacent regions.
Disease-oriented studies: identifies differentially modified mRNA sites as candidate biomarkers.
Method comparison: provides a quantitative reference when a conversion-based result needs to be compared with an enrichment-based profile of the same samples.

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: resuspend in ethanol or RNase-free ultrapure water, store at −80 °C and avoid repeated freeze–thaw cycles.

Shipping. Place the sample in a 1.5 mL RNase-free microcentrifuge tube, seal it with parafilm or a cap lock, and ship 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 10–300 µg total RNA.

08FAQ

What does a RedaC:T-seq result contain for each site?

A genomic coordinate, the host gene, the functional region the site falls in, and a modification ratio. The annotation is what lets a site be read in the context of its transcript.

Is an antibody used anywhere in this workflow?

No. Detection rests on a reductive chemical conversion, so no enrichment step and no antibody binding preference is involved.

How does this differ from an enrichment-based ac⁴C profile?

Enrichment-based methods report regions with elevated signal and carry the binding preference of the antibody used. RedaC:T-seq reports individual cytidines and a ratio for each, which is what a site-level comparison needs.

Can lncRNA be included?

Yes. Two scopes are available: mRNA alone, or mRNA together with long non-coding RNA. Both are analyzed in one experiment rather than split across two projects.

What research questions can a site-level ac⁴C map answer?

A site-level map supports questions about translation efficiency, transcript stability and condition-dependent acetylation on mRNA and long non-coding RNA. Positions that move between samples can be taken into focused validation without treating the mark as a bulk signal.

09References

Arango D, Sturgill D, Yang R, et al., Oberdoerffer S. Direct epitranscriptomic regulation of mammalian translation initiation through N4-acetylcytidine. Molecular Cell 2022;82(15):2797–2814.e11. DOI: 10.1016/j.molcel.2022.05.016

Single-Nucleotide RNA ac4C Modification Seq (RedaC:T-seq)