miCLIP-m5C Sequencing

m5C individual-nucleotide resolution crosslinking and immunoprecipitation sequencing (miCLIP-m5C-seq) is a next-generation sequencing (NGS)-based method to comprehensively detect m5C, a common and abundant RNA methylation modification that exists in various transcripts. Our experienced technical team provides you with comprehensive miCLIP-m5C-seq service and professional in-depth data analysis to satisfy customer needs.

Overview

m5C is one of the common and abundant RNA methylation modifications, mainly concentrated in variable arms, anti-codon loops, and regions where rRNA binds tRNA to perform translation activity. RNA m5C modification is also dynamically reversible. Methyltransferase transfers methyl group to cytosine C to form m5C with S-adenosine methionine (SAM) as the donor. As post-transcriptional methylation, m5C can affect different stages of mRNA metabolism, including genome imprinting, silencing of retrotransposons, suppression of transposition, repetitive sequence suppression, and X chromosome inactivation. Our company provides comprehensive miCLIP-m5C-seq service, which can accurately identify the full picture of m5C modification at single-nucleotide resolution level. In this technology, mRNA is cross-linked with m5C 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 m5C throughout the transcriptome. miCLIP-m5C-seq can accurately describe and quantitatively analyze m5C, providing an integrated perspective for understanding the growth and development, biological characteristics, and gene regulation mechanisms of organisms. In the aspect of disease research, it can be applied to studies of disease mechanism, and the screening of biomarkers.

Features

Single-nucleotide Resolution Application Transcriptome-wide High Efficiency
Maps m5C locations transcriptome-wide with single-nucleotide resolution. This method can profile and quantify m5C in small-RNA species and hm5C. Accurate localization of m5C 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.

Bioinformatics Analysis Pipeline

miCLIP-m5C Sequencing

In-depth data analysis:

  • Statistics of m5C distribution
  • Peaks annotation
  • Transcriptome-wide profiling of m5C methylation
  • Differential binding analysis
  • Motif search of enrichment sites
  • Evolutionary conservation analysis
  • Clustering analysis and enrichment analysis
  • Explore new m5C methylation sites
  • Identify m5C and m5C 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. Harry George, Jernej Ule, Shobbir Hussain, et al. Illustrating the Epitranscriptome at Nucleotide Resolution Using Methylation-iCLIP (miCLIP). Methods Mol Biol, 2017, 1562: 91-106. 
  2. Lindsey Van Haute, Sabine Dietmann, Laura Kremer, et al. Deficient Methylation and Formylation of mt-tRNA (Met) Wobble Cytosine in a Patient Carrying Mutations in NSUN3. Nat Commun, 2016, 6, 30(7): 12039.
  3. Shobbir Hussain, Abdulrahim A Sajini, Sandra Blanco, et al. NSun2-Mediated Cytosine-5 Methylation of Vault Noncoding RNA Determines Its Processing into Regulatory Small RNAs. Cell Rep, 2013, 7, 25, 4(2): 255-261.
* For Research Use Only. Not for use in diagnostic procedures.


Inquiry

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