PA-m6A Sequencing

Photo-crosslinking-assisted m6A sequencing (PA-m6A-seq) is a next-generation sequencing (NGS)-based method to comprehensively detect m6A. RNA methylation modification exists in various transcripts. It can map m6A in sample transcriptome with high precision. Our PA-m6A-seq service is dedicated to detecting and analyzing m6A RNA modification in a sample, providing a powerful tool for research of intracellular translation, regulation, and protein modifications.

Overview

N6-methyladenosine (m6A), methylation modification on the 6th nitrogen atom of RNA adenine, is one of the most abundant chemical modifications on eukaryotic mRNA. As a dynamic and reversible modification method, m6A is mainly enriched in the promoter region and termination codon region of mRNA. It plays an important role in regulating gene expression, splicing, RNA editing, RNA stability, controlling mRNA longevity and degradation, and mediating circular RNA translation is a hot spot in the epigenetic transcriptome, in which accurate identification of the m6A site will provide insights into its biological functions and mechanisms. We provide integrated PA-m6A sequencing service, which can map m6A in sample transcriptome with high precision. The technique synthesizes 4-thiouracil (4SU) into RNA by adding 4SU into the growth medium. After m6A immunoprecipitation, recovered m6A-containing RNA is cross-linked to the anti-m6A antibody under 365-nm ultraviolet light. Later, RNase T1 digests the cross-linked RNA to approximately 30nt and performing efficient sequencing. PA-m6A-seq at single-base resolution can determine the single consensus methylation sequences, including known and new m6A sites. In the meantime, this ultraviolet cross-linking strategy effectively provides insight into m6A-containing RNA and RNA-binding proteins.

Features

Single-base Resolution High Efficiency Professional Team Bioinformatics Service
Single-base resolution allows accurate detection of accurate sites of m6A. Adopts carefully optimized experimental procedure, achieving high efficiency and specificity. Provides various in-depth data analysis to satisfy customer needs. Provide standard and customized bioinformatics analyses, which are reflected in the report.

Project Workflow

Sample Preparation

1. Sample Preparation

RNA purification;
quality assessment and quantification.

Library Preparation

2. Library Preparation

RNA fragmentation; immunoprecipitation;
cross-linked reaction; m6A library preparation.

Sequencing

3. Sequencing

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

Data Analysis

4. Data Analysis

Visualize and preprocess results, and perform custom bioinformatics analysis.

PA-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

Sample Requirements

RNA sample quantity ≥ 300 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. Kai Chen, Zhike Lu, Xiao Wang, et al. High-Resolution N6-Methyladenosine (m6A) Map Using Photo-Crosslinking-Assisted m6A Sequencing. Angew Chem Int Ed Engl, 2015, 26, 54(5): 1587-1590.
  2. Caiyan Zhang, Jinrong Fu, Yufeng Zhou. A Review in Research Progress Concerning m6A Methylation and Immunoregulation. Front Immunol, 2019, 4, 26(10): 922.
  3. Xiaoyu Li, Xushen Xiong, Chengqi Yi. Epitranscriptome sequencing technologies: decoding RNA modifications. Nat Methods. 2016, 29, 14(1): 23-31.
* For Research Use Only. Not for use in diagnostic procedures.


Inquiry

  • Verification code
logo
Copyright © 2021 CD Genomics. All rights reserved.