hMeRIP Sequencing

Hydroxyl-methylcytosine RNA immunoprecipitation sequencing (hMeRIP-seq) is a next-generation sequencing (NGS)-based method to comprehensively detect 5-Hydroxymethylcytosine (hm5C), a new type of RNA methylation modification exists in the transcriptome. Our hMeRIP-seq service is dedicated to detecting the transcriptome-wide map of hm5c, fully revealing the distribution, location, and function of this RNA modification.

Overview

hm5c is one of RNA methylation exists in mammalian genomes, and is now generally regarded as the sixth base of genomic DNA. hm5C is generated by the oxidation of 5mC catalyzed by ten-eleven translocation enzymes and is known as an intermediate in demethylation via the TET-TDG pathway,  which has been predominantly observed in embryonic stem cells and neurons. In addition to mediating the process of DNA demethylation, hm5C is also an important epigenetic marker that can regulate gene expression. Furthermore, hm5C plays an important role in ovogenesis, cell differentiation, alternative splicing, and tumorigenesis. Our company provides comprehensive hMeRIP-seq service to fully reveal the intracellular distribution, location, and function of hm5c. hMeRIP sequencing is a high-throughput sequencing method based on antibody immunoprecipitation, in which RNA-protein complexes are cross-linked for stability, and antibodies specific to hm5C are added. As a new analysis method for detecting hm5C modification, hMeRIP-seq can comprehensively analyze the distribution and changes of hm5C in transcriptome, help researchers deeply understand the role of RNA modifications in life and disease, and promote accurate diagnosis and treatment of disease. At the same time, we provide one-stop hMeRIP-seq service to provide professional and in-depth data analysis for hm5c methylation to meet various needs of customers.

Features

Any Species High Efficiency Digitized Signal One-stop Service
This method can be applied to any species, from microorganisms to humans. Adopts carefully optimized experimental procedure, achieving high efficiency and specificity. Direct quantification and sequencing of methylated fragments without cross-reactivity and background noise. Provides one-stop service for library construction, sequencing, sample QC and data analysis.

Project Workflow

Sample Preparation

1. Sample Preparation

RNA purification; quality assessment and quantification.

Library Preparation

2. Library Preparation

RNA fragmentation; immunoprecipitation; cDNA 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

hMeRIP Sequencing

In-depth data analysis:

  • Peak calling and visualization
  • Peaks annotation
  • Biostatical analysis of hm5c data
  • Transcriptome-wide profiling of hm5c methylation
  • Differential binding analysis
  • Motif search of enrichment sites
  • Evolutionary conservation analysis
  • GO and KEGG pathway analysis

Sample Requirements

RNA sample quantity ≥ 50 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. Benjamin Delatte, Fei Wang, Long Vo Ngoc, et al. Transcriptome-wide Distribution and Function of RNA Hydroxymethylcytosine. Science, 2016, 1, 15, 351(6270): 282-285.
  2. Teng Zhang, Shao-Wu Zhang, Lin Zhang, et al. trumpet: transcriptome-guided quality assessment of m6A-seq data, BMC Bioinformatics, 2018, 7, 13, (19): 260.
  3. Lan A Roundtree, Molly E Evans, Tao Pan, et al. Dynamic RNA Modifications in Gene Expression Regulation, Cell, 2017, 8, 15(169): 1187-1192.
* For Research Use Only. Not for use in diagnostic procedures.


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