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MeRIP Sequencing (m6A-seq)

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The Introduction of MeRIP sequencing

RNA is subject to epigenetic modifications, and the post-translational modifications to RNA that can impact numerous biological processes have gained acceptance as a dynamic epigenetic mark. N6-methyladenosine (m6A) is the most common and abundant modification in messenger RNA and has been linked to diverse effects on mRNA fate. The in-depth study of its distribution and functions has revealed that m6A is typically near the stop codon, but also in the coding sequence, 3′UTR, and 5′UTR of mRNAs.

CD Genomics offers the transcriptome-wide analysis of the location of m6A in mRNA transcripts and other RNAs by methylated RNA immunoprecipitation sequencing (MeRIP-seq). MeRIPseq uses an antibody that specifically detects and characterizes of N6-methyladenosine (m6A) in RNA. As 3’UTR of mRNA plays important role in mRNA stability, localization, and translation, and may affect binding of RNA-regulatory proteins to these regions, enrichment of m6A in this region indicates that m6A may affect RNA metabolism and gene expression. Inhibition of the enzymes that involve in the RNA methylation has also provided clues to the potential biological roles of this modification. Hence, the identification and characterization of modified bases in RNA may lead to a better understanding of biological pathways.

MeRIP Sequencing Workflow

CD Genomics performs the m6A mapping approach using immunoprecipitation with m6A specific antibodies, followed by NGS (MeRIP-Seq). Poly(A)-selected RNA was fragmented into 100-nucleotide-long oligonucleotides (input) and immunoprecipitated using an anti-m6A affinity purified antibody and then subjected to Illumina next-generation sequencing. Because immunoprecipitated RNA fragments can contain m6A anywhere along their length, multiple different m6A-containing fragments generate overlapping reads. m6A sites were bioinformatically predicted by using a peak-detection algorithm and by searching for the presence of a subset of DRACH motifs near the point of highest read coverage.

MeRIP sequencing protocolFig. 1 MeRIP sequencing protocol

Service Specifications

Sample requirements

  • A minimum of 5 μg of total RNA is required. RIN value ≥ 7.0
  • Cells, tissue samples are accepted
Sequencing

Flexible service options include HiSeq 4000HiSeq X ten and NextSeq 500

Bioinformatics Analysis

  • Identification of m6A residues in RNA
  • The distribution of m6A peaks along transcripts
  • Differential peak analysis

Deliverables

  • The original sequencing data
  • Experimental results
  • Data analysis report
  • Details in MeRIP sequencing for your writing (customization)

CD Genomics’s MeRIP sequencing conference focuses on the links between cell variation in tissues and organ function and further elucidates the origins of diseases. If you have additional requirements or questions, please feel free to contact us.

Reference
Dominissini D, et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature, 2012 Apr 29; 485(7397):201-6.

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