AlkAniline-Seq reads two marks on tRNA at once. Heat under alkaline conditions takes the modified base off its ribose, aniline cuts the chain at the abasic site that is left behind, and the 5'-phosphate created at the next residue becomes the starting point of every read in the library.
Two marks, one cut, one library
The modified bases this assay reads share a chemical weakness: the bond between the base and the ribose is labile. m⁷G in the variable loop and m³C mostly in the anticodon loop both become abasic sites when the RNA is held under heat and alkaline conditions, so one treatment reaches both marks in their own structural context.
Arraystar Single-Nucleotide tRNA m⁷G & m³C Modification Seq(AlkAniline-Seq) is an end-to-end sample-to-data service for tRNA, from RNA sample QC and the cleavage chemistry 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.
Aniline cleavage sequencing is a base-resolution method in which the labile base of m⁷G or m³C is removed under heat and alkaline conditions, aniline cuts the tRNA backbone at the resulting abasic site, and the 5'-phosphate created at the next residue directs adapter ligation, so the cut itself reports the modified position.
Standard m⁷G and m³C project — custom designs and add-on analyses available on request
| Service Name | Price |
|---|---|
| Single-Nucleotide tRNA m⁷G & m³C Modification Seq(AlkAniline-Seq) |
Why a cut is a good coordinate
m⁷G and m³C sites are read together, so the two lists can be compared directly.
Each call is the nucleotide at which the chemistry stopped, not an enriched window over a tRNA locus.
The fraction of reads starting immediately 3′ of the cut is reported as a site level, so a position modified on part of the tRNA pool keeps an intermediate value.
The reaction decides which positions are called, and total RNA enters it as it is, so neither a binding preference nor a tRNA fractionation step stands between the sample and the tRNA map.
Sites carry relative coordinates, host tRNA gene, anticodon, isotype and structural-domain assignment.
RNA QC, cleavage chemistry, library construction, sequencing and analysis are handled as one project.
Why the cut can be trusted as a position
Cleavage at a modified base has been observed for decades; what makes it a sequencing method is turning the cut into a library. Under heat and alkaline conditions the modified base of m⁷G or m³C leaves its ribose and an abasic site remains, and aniline then breaks the backbone there by β-elimination, leaving a 5'-phosphate on the residue next to the modified base.
That phosphate is the positive selection. Only ends carrying it are ligated to adapters, so unmodified fragments are not subtracted as background - they never enter the library. After alignment the parental base separates the two lists, a cut read on a guanosine for m⁷G and a cut read on a cytidine for m³C, and the same treatment also reaches dihydrouridine and 5-hydroxycytidine where they occur.
From total RNA to an m⁷G and m³C site table
Total RNA passes sample QC and enters the cleavage chemistry directly: heat and alkali generate the abasic site, the RNA is then dephosphorylated, aniline cuts the chain, the new 5'-phosphate ends are ligated to sequencing adapters, and alignment and cut-position scoring produce the two site lists.
Bioinformatics for AlkAniline-Seq
Reads are mapped to a curated tRNA reference, cut positions are scored nucleotide by nucleotide, and the parental base separates m⁷G from m³C. Per-position levels are reported together with structural-domain and isotype annotation, distribution and differential results, and genome-browser tracks.
Where an m⁷G and m³C map fits
Asks whether an m⁷G change in a sample is accompanied by an m³C change, from the same preparation and the same run.
Relates a change at a tRNA position to ribosome occupancy and to the translation of the codons that tRNA family decodes.
Tests how METTL1/WDR4, METTL6 or METTL2A/2B perturbations shift each of the two site lists.
Compares site levels between tRNA isotypes and isoacceptors rather than across the tRNA pool as a whole.
Sets the cut-based map beside TRAC-seq or beside MeRIP-style enrichment for an independent line of evidence.
Sample handling and submission
| 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 10 µg total RNA. |
Submit at least 10 µg total RNA per sample; the amount includes material consumed by sample QC. Questions: support@arraystar.com
Questions we are asked about AlkAniline-Seq
Key references for m⁷G, m³C and aniline cleavage
Arraystar tRNA AlkAniline-Seq returns base-resolution m⁷G and m³C positions across mature tRNA — share your samples and we will scope the run.