miCLIP-seq
N6-methyladenosine, also called m6A, is a base modification behavior widely found on mRNA. The internal modification of mRNA is used to maintain the stability of mRNA. The current technical methods used to detect m6A include high-throughput sequencing, colorimetry, and LC-MS. Among them, LC-MS/MS and colorimetry can detect the overall m6A level of mRNA, while MeRIP-seq and miCLIP-seq(m6A individual-nucleotide-resolution cross-linking and immunoprecipitation combined with high-throughput sequencing) are high-throughput sequencing methods.
miCLIP-seq is to use the m6A antibody to enrich the m6A modification, and cooperate with the ultraviolet cross-linking technology to identify the m6A modification at the single-base level in the whole genome.
The process of miCLIP-seq:
- Fragment the enriched mRNA.
- Use immunomagnetic beads with m6A antibody to bind with m6A mRNA fragments.
- After using UV cross-linking for immunoprecipitation, connect a linker sequence to the 3'end of the mRNA fragment, and add a P32 radioactive label to the 5'end, and then transfer the membrane.
- After cutting the membrane and recovering according to the radioactive label, reverse transcription and purification and recovery of the mRNA fragments.
- Circularize the cDNA of the reverse transcriptome.
- Perform multiple linearization on the circularized cDNA, and then construct a sequencing library for computer sequencing.
Figure 1 The miCLIP protocol [1].
Compared with other m6A detection methods, the features of miCLIP are as follows:
- The m6A and m6A modifications can be accurately positioned, no nucleotide pretreatment is required, which saves experimental procedures.
- Based on feature mutation, modification can be accurately predicted.
- The experimental results have no offset, and the recognition is not affected by the peak shape.
- Single base resolution can be achieved.
As a very important RNA epigenetic modification, how m6A cooperates with DNA and histone epigenetics to regulate gene expression requires further exploration. miCLIP is a highly sensitive tool that may be widely used in other epigenetic modifications that occur on RNA.
Workflow of miCLIP-seq service in Profacgen:
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Reference
- Linder, Bastian, et al. "Single-nucleotide resolution mapping of m6A and m6Am throughout the tranome." Nature Methods 12.8 (2015):767.