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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:

  1. Fragment the enriched mRNA.
  2. Use immunomagnetic beads with m6A antibody to bind with m6A mRNA fragments.
  3. 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.
  4. After cutting the membrane and recovering according to the radioactive label, reverse transcription and purification and recovery of the mRNA fragments.
  5. Circularize the cDNA of the reverse transcriptome.
  6. Perform multiple linearization on the circularized cDNA, and then construct a sequencing library for computer sequencing.

 miCLIP-seq

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:

Workflow

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Reference

  1. Linder, Bastian, et al. "Single-nucleotide resolution mapping of m6A and m6Am throughout the tranome." Nature Methods 12.8 (2015):767.

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