miCLIP-m6A Sequencing

m6A individual-nucleotide resolution crosslinking and immunoprecipitation sequencing (miCLIP-m6A-seq) is a next-generation sequencing (NGS)-based method to comprehensively detect m6A, a common and abundant RNA methylation modification that exists in various transcripts. Our experienced technical team provides you with comprehensive miCLIP-m6A-seq service and professional in-depth data analysis to satisfy customer needs.

Overview

N6-methyladenosine (m6A) is a common and abundant RNA methylation modification, accounting for 0.1%~0.4% of all adenosine. The modification process is dynamic and reversible, and is coordinately regulated by methyl-transferase complex, demethylase and corresponding reader. As a post-transcriptional methylation, m6A can affect the different stages of mRNA metabolism and the production of long non-coding RNA and microRNA, as well as multiple biological processes, including neuronal development, cell transition, immune response, DNA damage response, and tumorigenesis. Therefore, accurate detection of m6A is of great significance for understanding its extensive relationship with human development and common diseases and has become an important research direction in the RNA field and epigenetic modification. We provide integrated miCLIP-m6A-seq service, which can accurately identify the complete picture of m6A modification. In this technology, mRNA is cross-linked with m6A antibody by ultraviolet light, and the antibody cross-linked RNA fragment is purified and converted into cDNA library. Later, the cDNA strand is circularized, re-linearized, amplified, and sequenced. Then, crosslink-induced mutations and truncations introduced during reverse transcription are analyzed to determine precise positions of m6A throughout the transcriptome. miCLIP-m6A-seq map m6A locations in the transcriptome at single-nucleotide resolution, effectively providing insight into m6A-containing RNA and RNA-binding proteins.

Features

Single- nucleotide Resolution Application Transcriptome-Wide High Efficiency
Maps m6A locations transcriptome-wide with single-nucleotide resolution. This method can profile and quantify m6A in small-RNA species and m6Am. Accurate localization of m6A sites in the global transcriptome. Adopts carefully optimized experimental procedure, achieving high efficiency and specificity.

Project Workflow

Sample Preparation

1. Sample Preparation

RNA purification;
quality assessment and quantification.

Library Preparation

2. Library Preparation

RNA fragmentation;
crosslinking reaction;
PCR amplification.

Sequencing

3. Sequencing

Illumina HiSeq;
PE 50/75/100/150.

Data Analysis

4. Data Analysis

Visualize and preprocess results, and perform custom bioinformatics analysis.

miCLIP-m6A Sequencing

Bioinformatics Analysis Pipeline

In-depth data analysis:

  • Statistics of m6A distribution
  • Peaks annotation
  • Transcriptome-wide profiling of m6A methylation
  • Differential binding analysis
  • Motif search of enrichment sites
  • Evolutionary conservation analysis
  • GO and KEGG pathway analysis
  • Explore new m6A methylation sites
  • Identify m6A and m6Am in small RNA

Sample Requirements

RNA sample quantity ≥ 200 ug.
Please make sure that the RNA is not significantly degraded.

Sample storage: RNA can be dissolved in ethanol or RNA-free ultra-pure water and stored at -80°C. RNA should avoid repeated freezing and thawing.

Shipping Method: When shipping RNA samples, the RNA sample is stored in a 1.5 mL Eppendorf tube, sealed with sealing film. Shipments are generally recommended to contain 5-10 pounds of dry ice per 24 hours.

Deliverable: FastQ, BAM, coverage summary, QC report, custom bioinformatics analysis.

References:

  1. Linder B, Grozhik A V, Olarerin-George A O, et al. Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome. Nat Methods, 2015, 8, 12(8): 767-772.
  2. Anya V G, Bastian L, Olarerin-George A O, et al. Mapping m6A at individual-nucleotide resolution using crosslinking and immunoprecipitation (miCLIP). Methods Mol Biol, 2017, 1562: 55-78.
  3. Wang X, Lu Z, Gomez A, et al. N6-methyladenosine-dependent regulation of messenger RNA stability. Nature, 2014, 505: 117-120.
  4. Maity A, Das B. N6-methyladenosine modification in mRNA: machinery, function and implications for health and diseases. FEBS J, 2016, 283:1607-1630.
* For Research Use Only. Not for use in diagnostic procedures.


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