tRNA m⁵C Methylation Sequencing

Chemical modification of nucleic acids is a key cellular process that occurs in all living organisms. As one of the commonly used RNA modifications, 5-Methylcytosine (m⁵C) presents in tRNA and rRNA molecules. m⁵C modification of RNA can affect the fate of the modified RNA molecule and plays an important role in a variety of biological processes, including RNA stability control, protein synthesis, and transcriptional regulation. Characterization of m⁵C methylation is therefore critical for developing the molecular basis of epigenetic gene regulation. In recent years, with the fast development of next-generation sequencing (NGS) technologies, scientists are continuously optimizing and improving the tRNA methylation sequencing methods. Combined with our stable and accurate sequencing tools, BOC Sciences provides tRNA m⁵C methylation sequencing services by offering reproducible and quantitative detection of cytosine methylation.

Fig 1. The distribution of methylation in mRNA. (Song H, 2022)

Application of m⁵C modification in tRNA

• Maintain the steady state
• Optimize codon-anticodon pairing
• Regulate stress response
• Control translation efficiency and accuracy

tRNA m⁵C Methylation Sequencing Workflow

• Sample Preparation: tRNA purification
• Library Construction: tRNA fragmentation and immune suppression
• Deep Sequencing
• Bioinformatics Data Analysis: Visualization processing results, coverage analysis, m⁵C-based global dynamics analysis, methylation level and density analysis, DMR annotation and enrichment analysis, clustering analysis

Fig 2. Principle of m⁵C detection by RNA bisulfite sequencing. (Motorin Y, 2009)

BOC Sciences tRNA m⁵C Methylation Sequencing Services

At BOC Sciences, we support a variety of tRNA m⁵C methylation detection technologies, which are available for the analysis of a large numbers of tRNA molecules.

• RNA BS-seq

RNA BS-seq has long been the most commonly used method for detecting methylated cytosines, providing single-nucleotide resolution and genome-wide coverage. In our RNA BS-seq strategy, sodium bisulfite is applied to convert un-methylated cytosines into uracil, while methylated cytosines remain unchanged. The cDNA is then amplified by PCR and we perform a deep sequencing of tRNA-derived cDNAs. Finally, m⁵C can be detected in tRNA by comparing untreated and sodium bisulfite-treated sequences. Our RNA BS-seq technical advantages include:

• High depth: ultra-high heavy bisulfite treatment provides high detection accuracy.
• High-throughput: Combined with high-throughput NGS, full transcriptome-wide detection can be achieved.
• Single base: Single base resolution for rapid detection and analysis of m⁵C in RNA.
• m⁵C-RIP-seq

As a complement to bisulfite sequencing services, our experts provide m⁵C-RIP-seq service to further confirm the methylation of target tRNAs. We use m⁵C-specific antibodies to enrich m⁵C-modified tRNAs from total tRNAs. Our sequencing service is highly specific and does not require the strict reaction conditions of bisulfite sequencing, and can be used to map m⁵C sites in low-abundance tRNAs.

• Aza-IP-seq

Aza-IP recognizes specific m⁵C sites on RNA m⁵C methyltransferase (RCMT) at a single nucleotide resolution. We provide Aza-IP-seq service to identify specific catalytic sites of RCMT.

• miCLIP-seq

In our well-established miCLIP-seq deep sequencing method, the m⁵C site is mapped at the +1 site of the sequencing read.

Advantages of Our tRNA m⁵C Methylation Sequencing Services

• Comprehensive investigation of the distribution and changes of m⁵C in transcriptome
• Un-methylated C conversion efficiency over 99%
• Deepen the understanding of the epigenome
• tRNA methylation patterns studies
• Study the biological function of RNA methylation.
• Provide accurate and sensitive maps of these methylation modifications
• Systematic search for m⁵C modifications with the application of both high-throughput sequencing methodologies and bioinformatics analysis

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tRNA m5C Methylation Sequencing

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