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Small RNA Modification Sequencing

m3C-IP-Seq — Single-Nucleotide tRNA m3C Modification Seq

m3C-IP-Seq profiles N3-methylcytidine on tRNA without antibodies. An immunoprecipitation step concentrates m3C-bearing fragments and a conversion read-out then registers the modified nucleotide at base resolution.

IP enrichment plus conversion read-out · antibody-free · per-site stoichiometry

Overview

Single-base m3C calling on tRNA

N3-methylcytidine sits on the Watson–Crick face, where it changes how cytidine pairs and how the anticodon loop behaves. Because it is present at a limited set of tRNA positions, mapping it means separating a small number of modified cytosines from the unmodified background.

Arraystar Single-Nucleotide tRNA m3C Modification Seq(m3C-IP-Seq) is an end-to-end sample-to-data service for tRNA, running from RNA sample QC and tRNA treatment through library construction, sequencing and bioinformatics. Sites are localized within mature tRNA transcripts with relative coordinates, gene attribution, anticodon and isotype, and structural-domain context.

What is tRNA m3C-IP-Seq?

m3C-IP-Seq is an antibody-free sequencing service in which immunoprecipitation enriches m3C-containing fragments and a conversion read-out records the modified nucleotide at single-base resolution, giving sensitive, quantitative m3C calling across tRNA.

Service at a Glance

Standard tRNA m3C project — custom designs and add-on analyses available on request

Service NamePrice
Single-Nucleotide tRNA m3C Modification Seq(m3C-IP-Seq)

Benefits

What m3C-IP-Seq adds to tRNA methylome work

Enrichment then resolution

The IP step concentrates m3C-bearing fragments, and the conversion read-out then fixes the modified nucleotide to a single base.

Quantitative fractions

Conversion signal at each position yields the m3C fraction, which can be compared between samples.

Antibody-free read-out

The calling chemistry avoids cross-reactivity, so specificity does not depend on antibody affinity.

Annotated within mature tRNA

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

Complementary to chemical cleavage

The workflow gives an independent route to tRNA m3C alongside cleavage-based sequencing.

One project, end to end

RNA QC, tRNA processing, library construction, sequencing and analysis are handled together.

Background

Reading m3C inside tRNA

m3C at the anticodon loop is a conserved tRNA modification, and its position determines how it affects structure and decoding. Distinguishing the modified cytidines from the many unmodified ones is the central analytical problem, particularly because tRNA is short and heavily folded.

Immunoprecipitation enriches m3C-containing fragments and a conversion read-out records the modified nucleotide at single-base resolution, giving sensitive m3C mapping inside mature tRNA transcripts. Arraystar runs this end to end and reports each site with its tRNA context (1).

tRNA m3C
Figure 1. m3C-IP-Seq detection scheme. An IP step enriches m3C-bearing fragments and a conversion read-out registers the modified nucleotide at base resolution.

tRNA m3C-IP-Seq Workflow

From total RNA to base-resolution m3C calls

The sample moves through RNA QC and tRNA treatment, m3C-directed enrichment, library construction and sequencing, and finally tRNA-aware calling that converts the read-out into per-site m3C values.

tRNA m3C-IP-Seq Workflow
Figure 2. Single-Nucleotide tRNA m3C Modification Seq workflow.

Bioinformatics & Deliverables

Bioinformatics for m3C-IP-Seq

Reads are mapped to a curated tRNA reference, m3C-IP-Seq conversion signatures are called, and per-position stoichiometry is reported with motif, differential, enrichment and genome-browser outputs.

Standard Deliverables

Research Applications

Research directions for tRNA m3C

Anticodon-loop function

Places m3C inside anticodon context to test its effect on decoding.

tRNA methylome comparison

Contrasts m3C occupancy between conditions or between tRNA families.

Writer and eraser studies

Tests how methyltransferase perturbations change m3C at specific sites.

Isotype-resolved analysis

Groups calls by isotype and isoacceptor for family-level comparisons.

Disease-oriented screening

Identifies differentially methylated tRNA positions between groups.

Sample Requirements

Sample handling and submission for tRNA m3C

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. Submit at least 200 µg total RNA.

Submit at least 200 µg total RNA per sample; this assay consumes more input than conversion-only workflows. Questions: support@arraystar.com

FAQ

Questions we are asked about tRNA m3C-IP-Seq

Why combine enrichment with a conversion read-out?
Immunoprecipitation concentrates the small fraction of tRNA fragments that carry m3C, and the conversion step then reports the modified nucleotide itself. Together they give sensitivity to rare sites while keeping base-level resolution, which neither step would deliver on its own.
Is the assay antibody-dependent in its read-out?
The enrichment step is targeted, but the calling chemistry is antibody-free and reproducible, so specificity comes from the conversion event rather than from antibody cross-reactivity. That keeps stoichiometry comparable between samples and reduces dependence on how efficiently an antibody reaches its target.
Which tRNA features are annotated?
Each site is reported with relative coordinates inside the mature tRNA transcript, host tRNA gene attribution, anticodon and isotype, and structural-domain localization, so a position can be interpreted within its own tRNA rather than as a coordinate on a generic reference.
How much total RNA does the assay require?
Around 200 µg of total RNA per sample is the working input, which is higher than conversion-only workflows because enrichment consumes material. Samples should pass QC with an OD260/280 ratio of 1.8 or higher and no visible degradation. Contact support to discuss lower-input options.
How does m3C-IP-Seq relate to cleavage-based m3C methods?
The two approaches reach the same modification by different chemistry. Running them in parallel provides independent confirmation of a site and lets cleavage-based and enrichment-based evidence be compared for the same tRNA, which is useful when a position is being reported for the first time.
Can differential m3C be tested between groups?
Yes. Per-position stoichiometry is reported for every sample, and differential analysis with fold change and significance is part of the standard pipeline, so m3C shifts between conditions can be called directly at the level of individual tRNA positions across a time course or treatment series.

Selected Publications

Key references for tRNA m3C

  1. Gao Y, Hou J, Wei S, et al. Transcriptome-wide mapping of N3-methylcytidine modification at single-base resolution. Nucleic Acids Research, 2025. PMID: 40071931

Map m3C Inside Your tRNA Samples

Arraystar tRNA m3C-IP-Seq combines targeted enrichment with a base-resolution read-out — send your sample set and we will return annotated, quantitative m3C calls.