N6-methyladenosine (m6A Individual-Nucleotide-Resolution Crosslinking and Immunoprecipitation
miCLIP-m6A maps m6A locations in the transcriptome with single-nucleotide resolution (Linder et al., 2015). In this method, anti-m6A antibodies are crosslinked to mRNA sequences, and a cDNA library is prepared and sequenced. The cDNA library preparation in miCLIP follows the iCLIP protocol closely.
Starting with total RNA, mRNA strands are isolated and fragmented. Anti-m6A antibodies are introduced and UV-crosslinked. The RNA-antibody complexes are immunoprecipitated and purified. Following the iCLIP cDNA library preparation protocol, the 3′ ends of the RNA are dephosphorylated and ligated to 3′ adapters. The RNA complexes are purified again before digesting the bound anti-m6A antibodies with proteinase K. The freed RNA strands are reverse-transcribed, and the resulting cDNA strands are circularized, relinearized, and PCR-amplified before sequencing. The m6A residues are identified accurately by analyzing the cDNA truncation and cytosine-to-thymine substitution patterns from the peptide residues on the RNA.
- Maps m6A locations transcriptome-wide with single-nucleotide resolution
- Identifies m6A in small-RNA species
- Able to identify m6Am in addition to m6A
- Does not involve pretreating cells with 4-SU, as in PAR-CLIP
- Dependent on the consistency of antibodies used in producing C-to-T substitution patterns
- cDNA library preparation uses radioactive labeling
Illumina Library prep and Array Kit Selector
Maity A. and Das B. N6-methyladenosine modification in mRNA: machinery, function and implications for health and diseases. FEBS J. 2016;283:1607-1630
Meyer K. D., Patil D. P., Zhou J., et al. 5′ UTR m(6)A Promotes Cap-Independent Translation. Cell. 2015;163:999-1010