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Small RNA Modification Sequencing

TRAC Seq — m7G — tRNA m7G Modification Seq at Single-Nucleotide Resolution

N7-methylguanosine (m7G) is a conserved tRNA modification predominantly located at position 46 in the variable loop of certain tRNAs, enzymatically deposited by the METTL1/WDR4 methyltransferase complex. Arraystar m7G TRAC-Seq maps m7G sites at nucleotide-level accuracy using highly specific chemical reactions, enabling quantitative stoichiometry assessment.

Chemical specificity · single-nucleotide resolution · quantitative stoichiometry

Overview

Chemical-based m7G detection without antibody affinity

m7G TRAC-Seq (tRNA reduction and cleavage sequencing) is a chemical method for single-nucleotide resolution profiling of m7G throughout the tRNA transcriptome. Isolated tRNAs are enzymatically demethylated and treated with NaBH₄/aniline to induce reductive cleavage specifically at m7G residues.

The 3′ cleavage fragments are ligated with a sequencing adaptor at their 5′-ends where the precise modification sites are located. The generated cDNA library for each sample is sequenced, and the sequencing data are analyzed for m7G sites and modification levels by cleavage scores.

What is m7G TRAC-Seq?

TRAC-Seq (tRNA reduction and cleavage sequencing) is a chemical method for single-nucleotide resolution profiling of m7G throughout the tRNA transcriptome. It relies on NaBH₄/aniline reductive cleavage at m7G residues, with demethylation controls, to identify m7G sites and modification levels by cleavage scores.

Service at a Glance

Standard m7G TRAC-Seq project — custom designs and add-on analyses available on request

Service NamePrice
m7G TRAC-Seq Service

Benefits

Key advantages of Arraystar m7G TRAC-Seq

🎯

Single-Nucleotide Resolution

Precise mapping of m7G sites at nucleotide-level accuracy.

⚗️

Chemical Specificity

Highly specific chemical reactions, instead of antibody affinity, to eliminate background from non-specific antibody binding and enable quantitative stoichiometry assessment.

🧬

Superior tRNA Coverage

Demethylation step using the Arraystar rtStar™ tRNA Pretreatment Kit to overcome tRNA modification barriers that block reverse transcription, allowing efficient sequencing of heavily modified tRNAs.

📊

Comprehensive Analyses

Simultaneous identification of m7G sites and discovery of modification-associated sequence motifs (e.g. RAGGU motif for m7G tRNAs).

🔗

Integrated Application

Works together with our tRF&tiRNA sequencing to uncover how m7G modification regulates tRF/tiRNA expression levels.

🖼️

Publication-Ready Visualization

Detailed bioinformatics analyses with volcano plots, read alignments, and motif analysis for m7G in tRNA biology, diseases, and biomarker applications.

Background

m7G in tRNA biology and disease

N7-methylguanosine (m7G) is a conserved tRNA modification predominantly located at position 46 in the variable loop of certain tRNAs, enzymatically deposited by the METTL1/WDR4 methyltransferase complex. It plays a pivotal role in maintaining tRNA structural integrity through formation of a C13–G22–m7G46 base triple, thereby shielding tRNAs from rapid decay pathways.

Functionally, m7G is indispensable for stem cell self-renewal and proper lineage commitment during embryonic development [1]. Moreover, m7G-modified tRNAs enhance the translation efficiency of codon-enriched mRNAs, particularly those encoding oncoproteins involved in cell cycle progression, proliferation, and growth signaling.

m7G dysregulation, especially with METTL1 overexpression, elevates levels of m7G-modified tRNAs (e.g. Arg-TCT-4-1, Lys-CTT, Val-AAC), driving tumorigenesis in diverse malignancies including leukemia, glioblastoma, cholangiocarcinoma, and lung cancer [2-4].

Figure 1. m7G TRAC-Seq workflow. Isolated tRNAs are enzymatically demethylated and treated with NaBH₄/aniline to induce reductive cleavage specifically at m7G residues. The 3′ cleavage fragments are ligated with a sequencing adaptor at their 5′-ends where the precise modification sites are located. The generated cDNA library for each sample is sequenced. The sequencing data are analyzed for m7G sites and modification levels by cleavage scores.
Figure 1. m7G TRAC-Seq workflow. Isolated tRNAs are enzymatically demethylated and treated with NaBH₄/aniline to induce reductive cleavage specifically at m7G residues. The 3′ cleavage fragments are ligated with a sequencing adaptor at their 5′-ends where the precise modification sites are located. The generated cDNA library for each sample is sequenced. The sequencing data are analyzed for m7G sites and modification levels by cleavage scores.

Background References

  1. Lin S, et al. Mettl1/Wdr4-Mediated m(7)G tRNA Methylome Is Required for Normal mRNA Translation and Embryonic Stem Cell Self-Renewal and Differentiation. Molecular Cell, 2018. PMID: 29983320
  2. Orellana EA, et al. METTL1-mediated m(7)G modification of Arg-TCT tRNA drives oncogenic transformation. Molecular Cell, 2021. PMID: 34352207
  3. Dai Z, et al. N(7)-Methylguanosine tRNA modification enhances oncogenic mRNA translation and promotes intrahepatic cholangiocarcinoma progression. Molecular Cell, 2021. PMID: 34352206
  4. Ma J, et al. METTL1/WDR4-mediated m(7)G tRNA modifications and m(7)G codon usage promote mRNA translation and lung cancer progression. Molecular Therapy, 2021. PMID: 34371184

m7G TRAC-Seq Workflow

From total RNA to single-nucleotide m7G maps

1

Sample QC

RNA quality and quantity assessment before the project proceeds, with small-RNA-retaining purification.

2

Demethylation

Isolated tRNAs are enzymatically demethylated to remove modification barriers, using the Arraystar rtStar™ tRNA Pretreatment Kit.

3

Reductive Cleavage & Library Construction

NaBH₄/aniline treatment induces reductive cleavage specifically at m7G residues; 3′ cleavage fragments are ligated with a sequencing adaptor at their 5′-ends.

4

High-Throughput Sequencing

The generated cDNA library for each sample is sequenced.

5

Bioinformatics

Sequencing data are analyzed for m7G sites and modification levels by cleavage scores, with motif analysis and differential analysis.

Bioinformatics & Deliverables

Detailed bioinformatics analyses included in m7G TRAC-Seq

The m7G TRAC-Seq includes detailed bioinformatics analyses to facilitate insights into m7G in tRNA biology, diseases, and biomarker applications.

Standard Deliverables

Figure 2. Volcano plot for differentially expressed m7G-tRNAs.
Figure 2. Volcano plot for differentially expressed m7G-tRNAs.
Figure 3. m7G TRAC-Seq read alignments for m7G site identification.
Figure 3. m7G TRAC-Seq read alignments for m7G site identification.
Figure 4. Motif analysis of m7G site identified by TRAC-Seq.
Figure 4. Motif analysis of m7G site identified by TRAC-Seq.

Research Applications

m7G tRNA modification research across biology and disease

Stem Cell Biology

m7G is indispensable for stem cell self-renewal and proper lineage commitment during embryonic development (Mol Cell, 2018).

Cancer Biology

METTL1 overexpression elevates m7G-modified tRNAs (Arg-TCT-4-1, Lys-CTT, Val-AAC), driving tumorigenesis in leukemia, glioblastoma, cholangiocarcinoma, and lung cancer (Mol Cell, 2021; Mol Ther, 2021).

Oncogenic Translation

m7G-modified tRNAs enhance translation efficiency of codon-enriched mRNAs encoding oncoproteins involved in cell cycle progression, proliferation, and growth signaling.

tRF/tiRNA Regulation

Combines with tRF&tiRNA sequencing to uncover how m7G modification regulates tRF/tiRNA expression levels.

Biomarker Discovery

Detailed bioinformatics support m7G studies in tRNA biology, diseases, and biomarker applications.

Sample Requirements

Official Arraystar sample submission requirements for m7G TRAC-Seq

RNA Amount & Quality

  • Total RNA input: > 5 µg per sample (official recommended minimum for the entire experiment in a single attempt, including sample QC). Supply twice the recommended minimum to avoid project delays.
  • Purification: TRIzol / RNA precipitation or an RNA isolation kit. Because tRNA is < 200 nt, use a kit specified to retain small RNAs (e.g. Qiagen miRNeasy).
  • Concentration: > 20 ng/µL by Nanodrop; OD260/280 ~2.0 (acceptable 1.7–2.1); OD260/230 > 1.8.
  • Integrity: sharp 18S/28S rRNA bands by gel, or RIN > 7.0 by Bioanalyzer (serum/plasma/exosome/FFPE RNA exempt).
  • DNase treatment: optional for gDNA removal; required if the sample is also used for qPCR.

Shipping Instructions

  • Ship RNA in nuclease-free water (> 20 ng/µL), freeze-dried, or in ethanol; store at −80 °C or in liquid nitrogen.
  • Use nuclease-free certified, screw-cap 1.5 mL microtubes; seal caps with Parafilm; place tubes in a plastic bag.
  • Use 10 kg dry ice as refrigerant; include a signed Project Form and the sample list.
  • Ship to: Arraystar Inc., 9430 Key West Avenue #128, Rockville, MD 20850, USA. Contact us before shipping.

FAQ

Common questions about m7G TRAC-Seq

How does TRAC-Seq detect m7G at single-nucleotide resolution?
TRAC-Seq (tRNA reduction and cleavage sequencing) is a chemical method for single-nucleotide resolution profiling of m7G throughout the tRNA transcriptome. Isolated tRNAs are demethylated and treated with NaBH₄/aniline to cleave specifically at m7G residues. 3′ cleavage fragments are ligated with an adaptor at their 5′-ends, where modification sites are located; m7G sites and levels are identified by cleavage scores.
Why use chemical reactions instead of antibodies for m7G detection?
The method relies on highly specific chemical reactions, instead of antibody affinity, to eliminate background from non-specific antibody binding and enable quantitative stoichiometry assessment. This provides precise mapping of m7G sites at nucleotide-level accuracy throughout the tRNA transcriptome. Chemical-based detection also avoids variability from antibody-based enrichment across batches.
What is the minimum amount of RNA required?
We recommend more than 5 µg of total RNA per sample — the official Arraystar recommended minimum for the entire experiment in a single attempt, including sample QC. Supplying twice the recommended minimum helps avoid project delays. RNA integrity must be preserved, because degraded RNA cannot be rescued by downstream steps.
How is tRNA coverage achieved for heavily modified tRNAs?
A demethylation step using the Arraystar rtStar™ tRNA Pretreatment Kit overcomes tRNA modification barriers that block reverse transcription, allowing efficient sequencing of heavily modified tRNAs. This ensures superior tRNA coverage for quantitative m7G profiling. The same pretreatment principle underlies Arraystar's optimized tRNA sequencing methodologies.
What bioinformatics analyses are included?
The m7G TRAC-Seq includes detailed bioinformatics analyses to facilitate insights into m7G in tRNA biology, diseases, and biomarker applications. Projects deliver m7G sites and modification levels by cleavage scores, volcano plots for differentially expressed m7G-tRNAs, read alignments for site identification, and motif analysis.
Can m7G TRAC-Seq be combined with tRF&tiRNA sequencing?
Yes. m7G TRAC-Seq works together with Arraystar tRF&tiRNA sequencing to uncover how m7G modification regulates tRF/tiRNA expression levels, linking tRNA modification status with the production of tRNA-derived small RNAs. Both services are sample-to-data offerings within Arraystar's broad tRNA research portfolio.

Selected Publications

Key References for m7G TRAC-Seq

  1. Lin S, et al. Mettl1/Wdr4-Mediated m(7)G tRNA Methylome Is Required for Normal mRNA Translation and Embryonic Stem Cell Self-Renewal and Differentiation. Molecular Cell, 2018. PMID: 29983320
  2. Orellana EA, et al. METTL1-mediated m(7)G modification of Arg-TCT tRNA drives oncogenic transformation. Molecular Cell, 2021. PMID: 34352207
  3. Dai Z, et al. N(7)-Methylguanosine tRNA modification enhances oncogenic mRNA translation and promotes intrahepatic cholangiocarcinoma progression. Molecular Cell, 2021. PMID: 34352206
  4. Ma J, et al. METTL1/WDR4-mediated m(7)G tRNA modifications and m(7)G codon usage promote mRNA translation and lung cancer progression. Molecular Therapy, 2021. PMID: 34371184

Ready to Map m7G Modifications at Single-Nucleotide Resolution?

Arraystar m7G TRAC-Seq combines chemical specificity with quantitative stoichiometry assessment — get a quote and a project timeline tailored to your study.