tRNA m⁷G Modification Sequencing

As a common RNA methylation modification, m⁷G modification refers to the modification of adding a methyl group to the 7th N-position of RNA guanine (G). A variety of clinical data suggests that m⁷G modification enables to affect tRNA function and many important cellular processes in living organisms, and m⁷G has become a new biomarker with important regulatory functions. Therefore, in order to further understand the role of this kind of modification in various physiological processes, scientists have applied various sequencing technologies to perform the accurate identification of the modification site. BOC Sciences' professional epitranscriptomics & sequencing experts and bioinformatics teams provide in-depth and comprehensive tRNA m⁷G methylation sequencing and data analysis services.

Fig 1. Positions of the m⁷G modification in tRNA. (Tomikawa C, 2018)

Our tRNA m⁷G Modification Sequencing Services

BOC Sciences provides a variety of m⁷G RNA methylation sequencing services:

• MeRIP-Seq

We provide MeRIP-Seq services by using pre-validated commercial antibodies and carefully optimized experimental procedures.

Our Sequencing Workflow

1. m⁷G-specific antibodies are employed to extract methylated fragments.
2. High-throughput sequencing and peak positioning for m⁷G modifications.
3. The m⁷G site contained in the RNA is converted into another new site that can generate reverse transcription base mutations.
4. The methylation level of the m⁷G site is estimated by evaluating the base mutation rate.

• TRAC-Seq

We offer a TRAC-Seq service for the unbiased global mapping of m⁷G modification of tRNAs. Our methods combine the selection of small tRNA molecules, AlkB demethylation, and sodium borohydride reduction steps to achieve specific and efficient single-nucleotide resolution analysis of m⁷G sites in tRNAs.

Our Sequencing Workflow

1. Modification Removal: We use the demethylase AlkB to remove major tRNA modifications.
2. tRNA Fragmentation: Sodium borohydride (NaBH₄) is used to reduce the m⁷G site, and we then conduct RNA chain cleavage using aniline for the generation of the abasic site.
3. Library Construction: The cleaved sites are then ligated with adapters for the libraries construction.
4. High-throughput Sequencing
5. Bioinformatics Pipeline: The identification of the m⁷G modification sites is completed by calculating the cleavage scores at individual sites on all tRNAs.

Fig 2. The formation of m⁷G modification. (Tomikawa C, 2018)

• m⁷G-Quant-Seq

BOC Sciences provides reliable m⁷G-quant-seq service, and our experts use chemometric information to detect abasic sites of m⁷G modifications on tRNA at single-base resolution. Through efficient chemical reduction and mild depurination, we can completely convert almost all m⁷G sites into RNA abasic sites.

Our Sequencing Workflow

1. Generation of abasic sites: Chemical structures of reduced m⁷G and RNA abasic site is generated by the treatment of KBH4-mediated reduction, followed by a depurination process.
2. Library construction: RNA fragmentation, RNA ligation, m⁷G-Quant-Seq and amplification & NGS sequencing.
3. Detection and quantification of internal m⁷G sites by measuring variation signals generated at AP site during reverse transcription.

BOC Sciences' Advantages

• High-throughput parallel detection of m⁷G sites in the whole transcriptome.
• One-stop service: RNA extraction, sample pretreatment, library construction, sequencing, and data analysis.
• In-depth data analysis.