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Small RNA Modification Sequencing

Single-Base tRNA Modification Sequencing — tRNA modification maps at single-nucleotide resolution

Twelve base-resolution assays map and quantify tRNA modifications at defined positions. Each assay reads the modification through chemistry, cleavage or a reverse-transcription signature, and every reported site carries its tRNA gene, isotype, anticodon and structural domain.

Twelve single-base tRNA assays · per-site modification fractions · antibody-free chemistry except m³C-IP-Seq

Overview

What the single-base tRNA assay family covers

Single-Base tRNA Modification Sequencing is Arraystar's assay family for reading tRNA modifications at single-nucleotide resolution. Sites are placed inside mature tRNA transcripts with relative coordinates, tRNA gene attribution, anticodon and isotype, and structural-domain localisation, so each result is a modification level at a defined position in a defined tRNA.

tRNA carries the densest set of modified nucleotides of any RNA class, and most of those modifications sit at conserved positions inside the folded L-shape. Position is therefore the informative unit: the same modification in the anticodon loop, the D-loop or the acceptor stem has a different meaning for decoding, folding and aminoacylation.

What is single-base tRNA modification sequencing?

It is a set of sequencing assays that convert, cleave or mis-incorporate at a modified tRNA nucleotide, so the modification is read at the base it occupies instead of being inferred from a region of enrichment. Each assay pairs its own chemistry with a tRNA-aware reference, which is what makes a position comparable between tRNA isotypes.

Benefits

Shared properties of the twelve assays

Base resolution

Each modification is assigned to a position in the mature tRNA transcript rather than to a tRNA population.

tRNA-aware annotation

Every site is reported with tRNA gene, isotype, anticodon and structural-domain context.

Antibody-free chemistry, with one exception

Conversion-, cleavage- and signature-based assays avoid antibody cross-reactivity and enrichment bias; m³C-IP-Seq uses m³C antibody enrichment.

Quantitative output

Conversion and signature read-outs report modification fractions; BACS-seq and m1A-Quant-seq report absolute stoichiometry.

Isotype-resolved analysis

Modification levels are compared across tRNA isotypes and isoacceptors on one reference.

Application-ready

Supports decoding fidelity, codon use, aminoacylation, tRNA structural biology and disease-associated modification changes.

Modifications Covered

Grouped by the nucleotide the modification sits on

Adenosine

  • m¹A (m1A-Quant-Seq)
  • m¹A, m¹G and m²,²G (tRNA Modification Seq)

Cytidine

  • m⁵C (BS-Seq)
  • m³C (m3C-IP-Seq, HAC-Seq)
  • ac⁴C (RedaC:T-Seq)

Guanosine

  • m⁷G (m7G-Quant-Seq, TRAC-Seq)

Uridine

  • pseudouridine Ψ (BID-Seq, BACS-Seq)
  • dihydrouridine D (CRACI-Seq)

Ribose

  • 2'-O-methylation Nm (NM-seq)

Assays at a Glance

Each assay name links to its service page, where chemistry, workflow, deliverables and input requirements are listed in full

m¹A · m1A-Quant-Seq

Engineered reverse transcriptase reads a reproducible mismatch at m¹A; reports an absolute fraction per position.

m¹A · m³C · m¹G · m²,²G · tRNA Modification Seq

Demethylase-comparison tRNA-seq calls four methylation types from one library at single-base resolution.

m⁵C · BS-Seq

Bisulfite conversion leaves m⁵C unconverted; the retained-C fraction gives the level per site.

m³C · m3C-IP-Seq

m³C-antibody enrichment ahead of a reverse-transcription misincorporation read-out; per-site stoichiometry.

m³C · HAC-Seq

Hydrazine-aniline cleavage cuts the backbone at m³C; sites are called by cleavage ratio.

ac⁴C · RedaC:T-Seq

Reductive conversion records acetylated cytidine as its own signature with a per-position fraction.

m⁷G · m7G-Quant-Seq

Targeted chemical conversion reports m⁷G as a fraction rather than a call.

m⁷G · TRAC-Seq

Reduction and cleavage sequencing locates m⁷G sites across the tRNA transcriptome by cleavage score.

Ψ · BID-Seq

Pseudouridine resists bisulfite conversion and appears as a deletion signature with a per-site fraction.

Ψ · BACS-Seq

Protection chemistry shields Ψ and reports it quantitatively to absolute stoichiometry.

D · CRACI-Seq

Conversion signature locates dihydrouridine to a single base inside the host tRNA.

Nm · NM-seq

Periodate protection marks 2'-O-methylated riboses that survive the oxidation cycles.

Workflow

The route from sample to single-base tRNA data is the same for the twelve assays

1. Assay confirmation

The target modification and the sample type are matched to the assay and to the input that assay needs.

2. Sample QC

RNA integrity and purity are checked before library construction, together with small-RNA-retaining purification for tRNA.

3. Library & sequencing

The assay-specific chemistry is applied and the library is sequenced on the Illumina platform.

4. Bioinformatics

Reads are mapped to a curated tRNA reference and single-base calls are reported with per-site levels and annotation.

Bioinformatics & Deliverables

Single-base tRNA calls with isotype, anticodon and structural-domain context

Downstream analysis maps reads to a curated tRNA reference, calls the conversion, cleavage or signature positions at single-base resolution, and reports modification levels per position with distribution, motif, differential and genome-browser outputs.

Standard Deliverables

The exact deliverable list is set per assay; the service page for each modification lists it in full.

Choosing Between Assays

Where more than one route exists, the chemistry decides

Ψ: two chemistries

BID-seq reads Ψ as a bisulfite-induced deletion signature, while BACS-seq protects Ψ and reports it quantitatively to absolute stoichiometry.

m³C: enrichment or cleavage

m3C-IP-Seq puts an m³C-specific antibody in front of a misincorporation read-out; HAC-Seq cleaves at m³C. Running both gives orthogonal confirmation of a site.

m⁷G: fraction or site map

m7G-Quant-Seq quantifies m⁷G as a fraction, and TRAC-Seq maps m⁷G sites across the tRNA transcriptome by cleavage score.

Modified cytidines

m⁵C BS-seq and ac⁴C RedaC:T-seq report different modifications through different chemistry, so the target mark decides which one applies.

Several methylation types at once

tRNA Modification Seq calls m¹A, m³C, m¹G and m²,²G from one library, which suits questions about how the tRNA methylome is rewired as a whole.

Expression and charging sit elsewhere

This family is about modification positions. tRNA abundance is covered by tRNA sequencing, and aminoacylation by tRNA Charging Seq.

Research Applications

Questions the family is built for

Decoding fidelity

Places modifications inside anticodon and wobble contexts to relate them to decoding.

Codon use & aminoacylation

Connects base-resolution modification to charging and translation for the same tRNA set.

tRNA structural biology

Localises sites in the D-loop, anticodon, TΨC arm and acceptor stem.

Isotype-resolved analysis

Compares modification levels across tRNA isotypes and isoacceptors.

Disease-oriented studies

Finds differentially modified tRNA sites as candidate biomarkers between conditions.

Orthogonal validation

Supports chemical confirmation of antibody-based tRNA modification results.

Sample Requirements

Material types shared by the family; the input amount is assay-specific

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 TypeNotes
Whole bloodUse EDTA tubes only; heparin is not compatible with subsequent analytical procedures.
Cultured cellsCell pellets are preferred to ensure high-quality material for processing.
TissueProvide fresh or frozen specimens and avoid necrotic material.
Total RNAMaintain an OD 260/280 ratio ≥ 1.8 and RIN ≥ 7 with no visible degradation.

Sample requirements are assay-specific: open the service page for the modification you need for the confirmed input amount. Questions: support@arraystar.com

FAQ

Questions we are asked about the single-base tRNA assays

Which tRNA modifications are covered by this family?
m⁷G, pseudouridine (Ψ), m⁵C, m¹A, m³C, 2'-O-methylation (Nm), dihydrouridine (D) and ac⁴C are read by the base-resolution assays, and tRNA Modification Seq adds m¹G and m²,²G to the same read-out. Twelve assays cover these marks because Ψ, m³C and m⁷G each have two chemistries.
How do I choose between the assays?
Start from the modification you need, then check the read-out on the assay card. Where a modification has two chemistries, the choice comes down to whether you want a site map, a per-site fraction, or orthogonal confirmation from a second route for the same position.
Are these assays antibody-based?
Most are not. The conversion-, cleavage- and signature-based assays read the modification chemically or enzymatically, while m³C-IP-Seq uses an m³C-specific antibody enrichment step ahead of its reverse-transcription misincorporation read-out and still reports single-base, per-site quantities.
Do you report tRNA position and isotype?
Yes. Sites are localised within mature tRNA transcripts with relative coordinates, tRNA gene attribution, anticodon and isotype, and structural-domain localisation, so a modification level can be read against the tRNA that carries it rather than as a coordinate on a generic reference.
Do the assays quantify, or only map?
Both, depending on the chemistry. Conversion- and signature-based read-outs report a modification fraction per site, and BACS-seq and m1A-Quant-seq report absolute fractions. Cleavage-based routes report sites with a cleavage score that is compared between samples or conditions.
Why does base resolution matter for tRNA?
tRNA is short and densely modified, and most modifications sit at conserved positions within the folded L-shape. A regional signal cannot separate those positions, so the position of a modification inside the mature transcript is what links it to decoding, folding or aminoacylation.

Selected Publications

Method papers behind the assays in this family

  1. Dai Q, Zhang LS, Sun HL, et al. Quantitative sequencing using BID-seq uncovers abundant pseudouridines in mammalian mRNA at base resolution. Nature Biotechnology, 2023;41(3):344-354. DOI: 10.1038/s41587-022-01505-w
  2. Xu H, Kong L, Cheng J, et al. Absolute quantitative and base-resolution sequencing reveals comprehensive landscape of pseudouridine across the human transcriptome. Nature Methods, 2024;21(11):2024-2033. DOI: 10.1038/s41592-024-02439-8
  3. Dai Q, Ye C, Irkliyenko I, et al. Ultrafast bisulfite sequencing detection of 5-methylcytosine in DNA and RNA. Nature Biotechnology, 2024;42(10):1559-1570. DOI: 10.1038/s41587-023-02034-w
  4. Li X, Xiong X, Zhang M, et al. Base-resolution mapping reveals distinct m1A methylome in nuclear- and mitochondrial-encoded transcripts. Molecular Cell, 2017;68(5):993-1005. DOI: 10.1016/j.molcel.2017.10.019
  5. Arango D, Sturgill D, Yang R, et al. Direct epitranscriptomic regulation of mammalian translation initiation through N4-acetylcytidine. Molecular Cell, 2022;82(15):2797-2814. DOI: 10.1016/j.molcel.2022.05.016
  6. Ju CW, Li H, Jiang B, et al. Quantitative CRACI reveals transcriptome-wide distribution of RNA dihydrouridine at base resolution. Nature Communications, 2025;16(1):8863. DOI: 10.1038/s41467-025-63918-w
  7. Cui J, Liu Q, Sendinc E, et al. Nucleotide resolution profiling of m3C RNA modification by HAC-seq. Nucleic Acids Research, 2021;49(5):e27. DOI: 10.1093/nar/gkaa1186
  8. Gao Y, Hou J, Wei S, et al. Transcriptome-wide mapping of N3-methylcytidine modification at single-base resolution. Nucleic Acids Research, 2025;53(5). DOI: 10.1093/nar/gkaf153
  9. Zhang LS, Ju CW, Liu C, et al. m7G-quant-seq: quantitative detection of RNA internal N7-methylguanosine. ACS Chemical Biology, 2022;17(12):3306-3312. DOI: 10.1021/acschembio.2c00792
  10. Dai Q, Moshitch-Moshkovitz S, Han D, et al. Nm-seq maps 2'-O-methylation sites in human mRNA with base precision. Nature Methods, 2017;14(7):695-698. DOI: 10.1038/nmeth.4294

Start From the Modification You Need

Open the assay that matches your target mark, or send the modification and the tRNA sample type you are working with and we will confirm which route applies — a site map, a per-site fraction, or both.