N3-methylcytidine (m³C) at position 32 of the anticodon loop is a conserved tRNA modification found in eukaryotic tRNASer, tRNAThr, and tRNAArg. Arraystar m³C HAC-Seq provides accurate and quantitative profiling of the tRNA m³C methylome for epitranscriptomics research.
Hydrazine-aniline cleavage sequencing for the tRNA m³C methylome
HAC-Seq (hydrazine-aniline cleavage sequencing) maps tRNA m³C modifications at single-nucleotide resolution. Isolated tRNAs are treated with hydrazine and aniline (HAC) to cleave the RNA backbone at m³C sites. To confirm specificity, samples are treated with demethylase prior to HAC (DM-HAC) to remove m³C modifications.
Since HAC-generated 5′ fragments contain damaged 3′ ends that preclude adapter ligation, sequencing captures only full-length and 3′ cleaved fragments. Consequently, m³C sites are identified at single-nucleotide resolution by calculating the cleavage ratio.
HAC-Seq (hydrazine-aniline cleavage sequencing) maps tRNA m³C modifications at single-nucleotide resolution. Hydrazine and aniline cleave the RNA backbone at m³C sites; demethylase-treated controls (DM-HAC) confirm specificity, and m³C sites are identified by calculating the cleavage ratio.
Standard m³C HAC-Seq project — custom designs and add-on analyses available on request
| Service Name | Price |
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| m³C HAC-Seq Service |
Key advantages of Arraystar m³C HAC-Seq
Provides precise mapping of m³C sites at nucleotide-level accuracy.
Relies on highly specific chemical reactions rather than antibody affinity, eliminating background from non-specific binding and enabling quantitative stoichiometry assessment.
Eliminates false positives by integrating m³C-demethylated samples as controls for significant cleavage induction and rescue.
Enables simultaneous identification of m³C sites and discovery of modification-associated sequence motifs.
Backed by Arraystar's expertise in tRNA research and the broad tRNA technology portfolio.
Detailed bioinformatics with IGV read alignments, sequence motif analysis, and clearly presented figures.
m³C in tRNA biology, translation, and disease
First identified in 1963, N3-methylcytidine (m³C) at position 32 of the anticodon loop is a conserved tRNA modification found in eukaryotic tRNASer, tRNAThr, and tRNAArg. This modification acts as a critical regulator of translation and cellular physiology.
In mitochondria, METTL8-dependent m³C on tRNAThr/Ser(UCN) is indispensable for protein synthesis, respiratory activity, and neural stem cell maintenance [1]. Cytoplasmic m³C32, mediated by METTL2A/2B/6 on tRNA-Ser-GCT, facilitates efficient AGU codon decoding and drives the translation of cell-cycle and DNA-repair regulators [2].
Beyond translation, m³C plays vital roles in disease and development: DALRD3-dependent modification of tRNA-Arg is crucial for neurological function [3], while METTL6-mediated modification of tRNA-Ser supports pluripotency and tumorigenesis [4]. Furthermore, nuclear METTL8 stabilizes R-loops via its methyltransferase activity, linking tRNA m³C modification machinery to genome organization [5].
From total RNA to single-nucleotide m³C maps
RNA quality and quantity assessment before the project proceeds, with small-RNA-retaining purification.
Isolated tRNAs are treated with hydrazine and aniline (HAC) to cleave the RNA backbone at m³C sites; parallel samples are treated with demethylase prior to HAC (DM-HAC) to remove m³C modifications and confirm specificity.
Because HAC-generated 5′ fragments contain damaged 3′ ends that preclude adapter ligation, sequencing captures only full-length and 3′ cleaved fragments.
High-throughput sequencing of the HAC and DM-HAC libraries.
m³C sites are identified at single-nucleotide resolution by calculating the cleavage ratio, with IGV read alignments and sequence motif analysis.
Detailed bioinformatics analyses included in m³C HAC-Seq
The m³C HAC-Seq provides a wealth of bioinformatics analyses to better understand their biology and facilitate biomarker applications.
m³C tRNA modification research across biology and disease
METTL8-dependent mitochondrial tRNA m³C is indispensable for protein synthesis, respiratory activity, and neural stem cell maintenance (Cell Stem Cell, 2023); DALRD3-dependent tRNA-Arg modification is crucial for neurological function (Nat Commun, 2020).
Cytoplasmic m³C32, mediated by METTL2A/2B/6 on tRNA-Ser-GCT, facilitates efficient AGU codon decoding and drives translation of cell-cycle and DNA-repair regulators (Nat Commun, 2024).
METTL6-mediated modification of tRNA-Ser supports pluripotency and tumorigenesis (Sci Adv, 2020); nuclear METTL8 stabilizes R-loops, linking m³C machinery to genome organization and tumorigenesis (iScience, 2020).
METTL8-dependent m³C on mitochondrial tRNAThr/Ser(UCN) supports respiratory activity and mitochondrial protein synthesis.
Detailed bioinformatics facilitate insights into m³C in tRNA biology, diseases, and biomarker applications.
Official Arraystar sample submission requirements for m³C HAC-Seq
Common questions about m³C HAC-Seq
Key References for m³C HAC-Seq
Arraystar m³C HAC-Seq combines chemical specificity with demethylase-controlled site identification — get a quote and a project timeline tailored to your study.