m3C-IP-Seq profiles N3-methylcytidine on tRNA with an m³C-specific antibody. Immunoprecipitation concentrates m³C-bearing fragments, and reverse-transcription misincorporation at the modified cytidine is then resolved to a single base.
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.
m3C-IP-Seq is an antibody-based sequencing service in which an m³C-specific antibody immunoprecipitates m³C-containing fragments, and reverse-transcription misincorporation at the modified cytidine is called at single-base resolution, giving sensitive, quantitative m³C profiling across tRNA.
Standard tRNA m³C project — custom designs and add-on analyses available on request
| Service Name | Price |
|---|---|
| Single-Nucleotide tRNA m³C Modification Seq(m3C-IP-Seq) |
What m3C-IP-Seq adds to tRNA methylome work
The antibody IP step concentrates m³C-bearing fragments, and the reverse-transcription read-out then fixes the modified nucleotide to a single base.
Misincorporation signal at each position yields the m³C fraction, which can be compared between samples.
An m³C-specific antibody concentrates the modified fragments, so low-abundance m³C positions are pulled out of the unmodified background.
Sites carry relative coordinates, host tRNA gene, anticodon, isotype and structural-domain assignment.
The workflow gives an independent route to tRNA m³C alongside cleavage-based sequencing.
RNA QC, tRNA processing, library construction, sequencing and analysis are handled together.
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.
An m³C-specific antibody immunoprecipitates m³C-containing fragments, and reverse-transcription misincorporation at the modified cytidine is resolved to a single base, giving sensitive m³C mapping inside mature tRNA transcripts. A matched AlkB demethylase (Dm) control removes m³C from one arm of the IP eluate, so true m³C sites show misincorporation only in the non-Dm arm and read back as unmodified C after demethylation. Arraystar runs this end to end and reports each site with its tRNA context (1).
From total RNA to base-resolution m³C calls
The sample moves through RNA QC and tRNA treatment, m³C-directed immunoprecipitation, a split of the IP eluate into an AlkB-demethylated arm (Dm) and an untreated arm (non-Dm), library construction and sequencing, and finally tRNA-aware calling. Reverse transcription records m³C as misincorporation in the non-Dm arm while the Dm arm reads as unmodified C; only sites that lose their signal after Dm are reported as high-confidence per-site m³C values.
Bioinformatics for m3C-IP-Seq
Reads are mapped to a curated tRNA reference, m3C-IP-Seq misincorporation signatures are called in the non-Dm arm, sites that persist in the AlkB-demethylated (Dm) arm are removed, and per-position stoichiometry is reported with motif, differential, enrichment and genome-browser outputs. The Dm versus non-Dm comparison turns raw misincorporation into high-confidence single-base calls.
Research directions for tRNA m³C
Places m³C inside anticodon context to test its effect on decoding.
Contrasts m³C occupancy between conditions or between tRNA families.
Tests how methyltransferase perturbations change m³C at specific sites.
Groups calls by isotype and isoacceptor for family-level comparisons.
Identifies differentially methylated tRNA positions between groups.
Sample handling and submission for tRNA m³C
| 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 | Maintain 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 the antibody-free chemical workflows. Questions: support@arraystar.com
Questions we are asked about tRNA m3C-IP-Seq
Key references for tRNA m³C
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.