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)
Fig 2. Principle of m5C detection by RNA bisulfite sequencing. (Motorin Y, 2009)
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 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:
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 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.
In our well-established miCLIP-seq deep sequencing method, the m5C site is mapped at the +1 site of the sequencing read.
References
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