Single-Nucleotide RNA m⁷G Modification Seq (m7G-Quant-seq)

01What the service reports

Single-Nucleotide RNA m⁷G Modification Seq (m7G-Quant-seq) is a base-resolution method in which a targeted chemical conversion distinguishes internal N⁷-methylguanosine from the 5′ cap, so internal sites can be detected and quantified rather than read together with the cap.

The output is a site table across protein-coding transcripts, each entry carrying a genomic coordinate, host gene, functional region and a modification ratio. Because the discrimination is chemical rather than antibody-based, no binding preference is imposed on which sites are seen.

Arraystar delivers m7G-Quant-seq as a sample-to-data service: RNA QC, the targeted conversion, library construction, sequencing and bioinformatics. Two transcript scopes are offered, so a study can profile mRNA alone or mRNA together with long non-coding RNA in one experiment.

02Benefits

• Internal sites separated from the cap: the conversion distinguishes internal m⁷G from the 5′ cap, which is the distinction that otherwise blurs the signal.
• Single-base resolution: every internal site is recorded as its own reverse-transcription signature.
• Site-level stoichiometry: the conversion-based readout gives a modification ratio per site.
• Antibody-free: no enrichment step, so antibody cross-reactivity and pull-down bias are not sources of variation in this assay.
• Annotated output: sites are mapped to the reference transcriptome with coordinates, host gene and functional region.

03Background and detection principle

N⁷-methylguanosine occurs both at the 5′ cap and internally, and the two populations behave differently in a transcript. Studies of internal m⁷G therefore need the cap signal removed rather than averaged in, and they need positions rather than a bulk level.

m7G-Quant-seq separates the two by chemistry. A targeted chemical conversion reacts with internal m⁷G and leaves a distinct reverse-transcription signature, while the cap is handled differently in the same chemistry. That makes the internal sites callable and quantifiable at base resolution, without an antibody and without a motif preference being imposed on the result. The workflow extends to lncRNA within the same experiment when the wider scope is selected.

Internal m⁷G becomes measurable once the cap is taken out of the same signal.m7G-quant-seq: quantitative detection of RNA internal N7-methylguanosine, ACS Chemical Biology 2022;17(12) · DOI 10.1021/acschembio.2c00792

04Workflow

Five steps take a sample from RNA to a single-base internal m⁷G table:

1. RNA sample QC, covering integrity and purity.
2. Targeted chemical conversion of the RNA.
3. Library construction from the converted material.
4. Sequencing of the m7G-Quant-seq library.
5. Bioinformatics: signature calling, internal-site annotation and stoichiometry estimation.

05Bioinformatics and deliverables

Reads are aligned to the reference transcriptome, m7G-Quant-seq conversion signatures are recognized, and internal m⁷G stoichiometry is estimated per site. The analysis set covers motif characterisation, transcript distribution, differential modification, functional enrichment and genome-browser visualization.

Deliverables

• Raw m⁷G mRNA sequencing data files (FASTQ), formatted for GEO/SRA submission.
• Sample and sequencing QC reports, sorted BAM alignment files and core mapping statistics.
• High-confidence single-base internal m⁷G site tables (XLSX) with coordinates, host gene and stoichiometry.
• Single-base m⁷G transcript feature distribution figures and consensus motif logos (PDF/PNG).
• Single-base differential m⁷G tables (XLSX) with fold change and significance.
• GO enrichment reports for m⁷G-modified genes (HTML/PDF/PNG).
• Genome-browser track files (bigWig/bedGraph) and a structured project report.

06Research applications

• Internal versus cap: separates the internal m⁷G population from the cap so the two are not reported as one number.
• Transcriptome-wide mapping: resolves internal m⁷G sites across protein-coding transcripts, and across lncRNA when that scope is selected.
• Translational control: connects a resolved internal site to mRNA translation and stability.
• Coding-region function: localises sites within CDS, UTR and splice-adjacent regions.
• Disease-oriented studies: identifies differentially modified sites as candidate biomarkers.

07Sample requirements

Storage. Cells and tissues: preserve in TRIzol or an RNA stabilisation solution, snap freeze in liquid nitrogen and store at −80 °C. RNA: resuspend in ethanol or RNase-free ultrapure water, store at −80 °C and avoid repeated freeze–thaw cycles.

Shipping. Place the sample in a 1.5 mL RNase-free microcentrifuge tube, seal it with parafilm or a cap lock, and ship on dry ice with adequate insulation.

08FAQ

09References

1. m7G-quant-seq: quantitative detection of RNA internal N7-methylguanosine. ACS Chemical Biology 2022;17(12). DOI: 10.1021/acschembio.2c00792 PMID: 36398936.