tRNA ac4C RedaC:T-seq | Single-Nucleotide ac4C Profiling on tRNA

Overview

Single-base ac4C quantification on tRNA

ac4C sits on the Watson–Crick face of cytidine, where it alters how the base pairs and how the anticodon loop behaves. Locating it base by base separates ac4C-bearing positions from unmodified cytidine, which is what makes it possible to link the mark to decoding and to tRNA stability.

Arraystar Single-Nucleotide tRNA ac4C Modification Seq(RedaC:T-seq) runs the complete workflow for tRNA: RNA quality control, tRNA processing, reductive handling, library preparation, sequencing and bioinformatics. Detected sites are placed inside mature tRNA transcripts and annotated by anticodon, isotype and structural domain.

What is tRNA RedaC:T-seq?

RedaC:T-seq is an antibody-free, base-resolution method for N4-acetylcytidine on tRNA. A reductive step converts acetylated cytidine into a distinct read-out, so ac4C is separated from unmodified cytidine and each site is called and quantified at single-nucleotide resolution.

Service at a Glance

Standard tRNA ac4C project — custom designs and add-on analyses available on request

Service Name	Price
Single-Nucleotide tRNA ac4C Modification Seq(RedaC:T-seq)

Benefits

Why teams use tRNA RedaC:T-seq

Ac4C fixed to one nucleotide

The reductive read-out separates acetylated from unmodified cytidine, so each site is resolved rather than averaged over a region.

Stoichiometry from the read-out

The proportion of converted signal at a position yields the ac4C fraction, which supports quantitative comparison between conditions.

No enrichment step

Antibody-free handling removes pull-down bias and cross-reactivity, which matters inside compact and heavily folded tRNA.

Annotated within mature tRNA

Sites carry relative coordinates, host tRNA gene, anticodon, isotype and structural-domain assignment.

Isotype and anticodon context

Positions can be grouped by isotype and anticodon so that decoding-related questions can be asked directly.

End-to-end execution

QC, tRNA processing, library construction, sequencing and analysis are handled as one project.

Background

Ac4C, pairing chemistry and tRNA decoding

N4-acetylcytidine changes the base-pairing behaviour of cytidine and has been placed at the centre of translation control. On tRNA it sits where decoding and structural stability are decided, so a position-level map is needed before its contribution to a specific isotype can be tested (1).

Reductive conversion acts on the acetylated base and records it as a readable signature while leaving unmodified cytidine distinguishable. Arraystar applies that chemistry in a tRNA workflow, then anchors each call to the mature transcript, its gene and its structural domain (1).

tRNA ac4C RedaC:T-Seq Workflow

From total RNA to base-resolution ac4C calls

The project moves in a single line: RNA QC with small-RNA-retaining purification, tRNA processing and reductive labelling, library construction and sequencing, then tRNA-aware calling of ac4C positions.

tRNA ac4C RedaC:T-Seq Workflow — Figure 2. Single-Nucleotide tRNA ac4C Modification Seq workflow.

Bioinformatics & Deliverables

Bioinformatics for RedaC:T-seq

Reads are aligned to a curated tRNA reference and reductive conversion signatures are called as ac4C positions. The pipeline returns per-position stoichiometry together with motif analysis, differential modification testing and browser-ready tracks.

Standard Deliverables

Raw sequencing reads (FASTQ) formatted for GEO/SRA deposition.
QC report with sorted BAM files and core mapping statistics.
Single-base ac4C site tables (XLSX) with coordinates, anticodon, isotype and stoichiometry.
ac4C distribution and motif figures across tRNA domains (PDF/PNG).
Differential ac4C tables (XLSX) with fold change and significance.
Genome-browser tracks (bigWig/bedGraph) with a structured tRNA report.

Research Applications

Research directions for tRNA ac4C

Translation initiation and decoding

Links ac4C positions to decoding behaviour and to translation output.

tRNA structural stability

Tests whether the mark at a given domain changes folding or turnover.

Wobble-position questions

Places ac4C inside anticodon and wobble context for base-pairing studies.

Isotype-resolved comparison

Compares ac4C occupancy between tRNA isotypes and isoacceptors.

Disease-oriented screening

Identifies differentially acetylated tRNA positions between groups.

Sample Requirements

Sample handling and submission for tRNA ac4C

Sample Storage

For cells/tissue: use TRIzol or an RNA-stabilizing reagent, quick-freeze in liquid nitrogen, and keep at –80 °C.
For RNA: dissolve in ethanol or RNase-free water, store at –80 °C, and limit freeze–thaw cycles.

Shipping Instructions

Transfer each sample into a 1.5 mL nuclease-free tube.
Close the tube securely with parafilm or a cap lock to preserve integrity.
Send on dry ice with sufficient insulation to sustain the required temperature.

Sample Type	Notes
Whole blood	Use EDTA tubes only; heparin is not compatible with subsequent analytical procedures.
Cultured cells	Cell pellets are preferred to ensure high-quality material for processing.
Tissue	Provide fresh or frozen specimens and avoid necrotic material.
Total RNA	Ensure OD260/280 ≥ 1.8, RIN ≥ 7 and no visible degradation; submit ≥ 10–300 µg total RNA.

Submit at least 10–300 µg total RNA per sample, depending on the number of target sites and the sample type. Questions: support@arraystar.com

FAQ

Questions we are asked about tRNA ac4C profiling

Does RedaC:T-seq read ac4C at single-base resolution?

Yes. The reductive step acts specifically on acetylated cytidine and records it as a distinct signature while unmodified cytidine reads differently, so each ac4C position is called at single-nucleotide resolution and quantified from the converted fraction rather than from regional enrichment.

Why avoid antibody enrichment for tRNA?

Enrichment-based methods resolve regions rather than positions and show motif bias, and the compact folding of tRNA restricts antibody access. RedaC:T-seq reads chemistry instead, so calling is quantitative at the site level and does not depend on pull-down efficiency or on how well an antibody reaches its target.

Which tRNA features are annotated?

Each site is placed inside a mature tRNA transcript with relative coordinates, host tRNA gene attribution, anticodon and isotype, and structural-domain localization, so a position can be interpreted within its own tRNA rather than as an unlabelled coordinate on a reference.

How much RNA should be submitted?

Between 10 and 300 µg of total RNA per sample is the working range, with the amount depending on sample type and the number of target sites. Samples should pass QC with an OD260/280 ratio of 1.8 or higher and no visible degradation.

Can ac4C be compared between isotypes?

Yes. Because isotype and anticodon are attached to every call, occupancy can be compared between isotypes and isoacceptors, and differential modification can be tested between sample groups on the same annotation, which keeps comparisons between isotypes internally consistent across the whole project.

How does ac4C profiling relate to translation studies?

ac4C sits on the base-pairing face of cytidine, so a position-level map tells you where in a tRNA the mark sits relative to decoding. That is the level of detail needed when ac4C is being linked to codon usage, translation initiation or tRNA turnover across a sample series.

Selected Publications

Key references for tRNA ac4C

Arango D, Sturgill D, Yang R, et al. Direct epitranscriptomic regulation of mammalian translation initiation through N4-acetylcytidine. Molecular Cell, 2022. PMID: 35679869

RedaC:T-Seq — Single-Nucleotide tRNA ac4C Modification Seq