MeRIP-seq Service

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MeRIP-seq Service

IntegrateRNA offers quick, efficient and precise identification of m6A modification site in mRNA transcripts and other RNAs by methylated RNA immunoprecipitation sequencing (MeRIP-seq). Combining Co-immunoprecipitation with high-throughput sequencing, m6A peak and m6A motif can be systematically studied within the transcriptome.

RNA m6A methylation is a post-transcriptional modification that occurs at the nitrogen-6 position of adenine. This dynamically reversible modification is installed, removed and recognized by methyltransferases, demethylases and readers, respectively. This modification has been found in most eukaryotic mRNA, tRNA, rRNA and other non-coding RNA. RNA m6A methylation can functionally regulate the eukaryotic transcriptome to affect the splicing and output of mRNA nuclear, positioning, translation and stabilization. The appearance of m6A RNA in a variety of important cell life processes means that mRNA methylation is involved in a variety of biological processes, such as stem cell differentiation, biological rhythms, disease development, etc. Thus, an unbiased method for transcriptome-wide localization of m6A may lead to a better understanding of biological pathways.

Workflow of MeRIP-seq

IntegrateRNA utilizes a highly m6A-specific antibody to immunoprecipitate methylated RNA fragments out of a randomly fragmented transcriptome, followed by NGS. Firstly, purified RNA is chemically fragmented into ~100-nt-long oligonucleotides and subjected to immunoprecipitation using an m6A-specific antibody. Eluted m6A-containing fragments (IP) and untreated input control fragments are converted to cDNA using random hexamer primers followed by adapter ligation and high-throughput sequencing (Figure 1). Finally, Bioinformatics analysis like peak calling and visualization, peak annotation, motif analysis and differential methylation analysis are performed using IP and input control data.

Schematic diagram of the MeRIP-seq protocolFigure 1. Schematic diagram of the MeRIP-seq protocol

Bioinformatics Analysis Pipeline

MeRIP-seq Service

In-depth Data Analysis Items

  • Genome mapping and functional annotation of all mapped reads
  • m6A peak calling and visualization in whole transcriptome
  • Motif analysis of m6A peak sequences
  • Differential methylation analysis
  • GO/KEGG enrichment analysis of differential gene/peak
  • Custom-tailored data analysis

Sample Requirement

  • Total RNA: >20ug (conc: >200ng/ul; RIN≥6.5; OD260/280=1.8~2.2; OD260/230≥2.0)
  • Cell: >10*7
  • Tissue: >100mg (Cut large tissue samples to ≤ 0.2cm in any single dimension. Plate three pieces of tissue in 1ml RNAlater solution.)

IntegrateRNA m6A-seq profiles the most prominent epitranscriptomic m6A modification by methylated RNA immunoprecipitation sequencing (MeRIP-seq). If you have additional requirements or questions, please feel free to contact us.

Reference:

  1. Dominissini, D. et al. (2013). Transcriptome-wide mapping of N6-methyladenosine by m(6)a-seq based on immunocapturing and massively parallel sequencing. Nature Protocols, 8(1), 176-189.
For research use only. Not intended for any clinical use.
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