m5C-RIP Sequencing

5-methylcytosine-RNA Immunoprecipitation (m5C-RIP) sequencing is a next generation sequencing (NGS)-based method to comprehensively detect m5C, one type of the most common and abundant methylation modifications in transcriptome. It can accurately explore the mechanisms of intercellular communication and biomarkers for disease monitoring. We provide high-throughput technology for sequencing m5C, which can quickly and efficiently obtain global information about RNA modification.

Overview

5-Methylcytosine (m5C) is one of the longest known post-transcriptional RNA modifications, and is mediated by the DNMT2 and NSUN methyltransferase family. It is highly popular in mRNAs and some non-coding RNAs of prokaryotes and eukaryotes. m5C modification is concentrated in CG-rich regions and regions immediately downstream of translation initiation sites and has conversed, and has the characteristics of conservation, tissue-specific and dynamic features across mammalian transcriptomes. Studies have shown that 5mC methylation modification plays a significant role in many biological processes such as genome imprinting, silencing of retrotransposons, suppression of transposition, repetitive sequence suppression, and X chromosome inactivation. Here, our company provide mature and advanced m5C-RIP-seq service, which can quickly identify the full picture of m5C modifications in various RNA molecules. The procedure includes RNA extraction, fragmentation, RNA immunoprecipitation, and library construction. Based on the principle of antibody-specific binding, the technology uses m5C-specific antibodies to enrich m5C-modified mRNAs from total mRNAs, and combines high-throughput sequencing technology and bioinformatics analysis to facilitate the genome-wide systematic search on the modification of m5C. m5C-RIP-seq has higher specificity, improves the detection ability of RNA modification, and can accurately identify m5C sites. It allows researchers to further explore epigenetics.

Features

High Coverage High Efficiency Rich Experience Strict Quality Control
The method can comprehensively detects m5C sites of multiple RNA molecules. Adopts carefully optimized experimental procedure, achieving high efficiency and specificity. Provide professional bioinformatics analysis team to meet comprehensive needs of customers. Quality control at every stage from library construction to bioinformatics analysis.

Project Workflow

Sample Preparation

1. Sample Preparation

RNA purification;
quality assessment and quantification.

Library Preparation

2. Library Preparation

RNA fragmentation;
Immunoprecipitation;
m5C 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.

Bioinformatics Analysis Pipeline

m5C-RIP Sequencing

In-depth data analysis:

  • Sequencing depth and coverage analysis
  • Peak calling and visulization
  • Transcriptome-wide profiling of m5C methylation
  • Differential binding analysis
  • Motif search of enrichment sites
  • Methylation level and density analysis
  • Clustering analysis and enrichment analysis

Sample Requirements

RNA sample quantity ≥ 50 ug.
1.6 ≤ OD260/280 ≤ 2.3, 28S:18S ≥ 1.5, RIN ≥ 7.
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. Cui X, Liang Z, Shen L, et al. 5-Methylcytosine RNA Methylation in Arabidopsis Thaliana. Molecular Plant, 2017, 10(11).
  2. Xin Yang, Ying Yang, Bao-Fa Sun, et al. 5-methylcytosine promotes mRNA export-NSUN2 as the methyltransferase and ALYREF as an m5C reader. Cell Research, 2017, 27: 606-625.
  3. Shobbir Hussain, Jelena Aleksic, Sandra Blanco, et al. Characterizing 5-methylcytosine in the mammalian epitranscriptome. Genome Biology, 2013, 14:215.
* For Research Use Only. Not for use in diagnostic procedures.


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