m7G+m3C Sequencing

RNA modifications play important roles in gene expression regulation. CD Genomics provides m7G + m3C sequencing service for the simultaneous identifications of 7-methylguanosine (m7G) and 3-methylcytidine (m3C) in RNA including both known and previously unmapped positions at single nucleotide resolution.

Overview

Chemical RNA modifications as central features of epitranscriptomics are highlighted by the discovery of modified ribonucleosides in mRNA and non-coding RNAs and exemplified by the vital roles of RNA modifications in normal physiology and disease. Methylation of guanosine on position N7 (m7G) on RNA has been found on various types of RNA molecules including mRNA 5’ cap structure, internal mRNA regions, pri-miRNA, tRNA and rRNA. m7G modifications play critical roles in mRNA transcription, miRNA biogenesis and functions, tRNA stability, and 18S rRNA maturation. The m7G RNA methylation has become a hot research topic in recent years. The m3C RNA methylation has been discovered on mRNAs, tRNAs, and rRNAs, however, the biochemistry of m3C formation in mammalian RNA and its functions are still poorly understood. To expand the list of NGS-detectable RNA modifications, CD Genomics has developed multiple products targeting m7G/m3C methylation. We also provide one-stop m7G+m3C RNA methylation sequencing services, simultaneously detecting global m7G and m3C methylations present in mRNA, lncRNA, pri-miRNA, tRNA, rRNA and other RNA molecules at single base resolution. Additionally, we provide m7G MeRIP-Seq for profiling global m7G modification sites. CD Genomics will gladly support your epitranscriptomics projects with challenging or unusual requirements.

Features

Superb Expertise Transcriptome-Wide High Accuracy One-Stop Solution
Decade of experience in epitranscriptomic sequencing and bioinformatics analysis. Profile global m7G/m3C sites on mRNA, lncRNA, pri-miRNA, tRNA,,and rRNA. Stringent QC with PCR; calibrated m7G/m3C levels; single-base resolution. One-stop solution from sample QC and library construction, to sequencing and data analysis.

Project Workflow

Sample Preparation

1. Sample Preparation

RNA purification;
quality assessment and quantification

Library Preparation

2. Library Preparation

RNA fragmentation;
m7G/m3C 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

m7G+m3C Sequencing

In-depth data analysis:

  • Peak calling and visualization
  • Peaks annotation
  • Transcriptome-wide profiling of m7G/m3C methylation
  • Differential binding analysis
  • Motif search of enrichment sites
  • Evolutionary conservation analysis
  • Gene ontology and KEGG enrichment analysis

Sample Requirements

RNA sample: quantity ≥ 300 ug.

1.8 ≤ OD260/280 ≤ 2.2, OD260/230 ≥ 2.0, RIN ≥ 6.5, 28S:18S ≥ 1.0.

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. Marchand V, Ayadi L, Ernst F G M, et al. AlkAniline‐Seq: Profiling of m7G and m3C RNA Modifications at Single Nucleotide Resolution. Angewandte Chemie International Edition, 2018, 57(51): 16785-16790.
  2. Xu L, Liu X, Sheng N, et al. Three distinct 3-methylcytidine (m3C) methyltransferases modify tRNA and mRNA in mice and humans. Journal of Biological Chemistry, 2017, 292(35): 14695-14703.
* For Research Use Only. Not for use in diagnostic procedures.


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