RNA Bisulfite Sequencing

RNA bisulfite sequencing (RNA BS-seq) is a next-generation sequencing (NGS)-based to accurately detect m5C, DNA methylation extensively exists in the human transcriptome. Our RNA BS-seq service is dedicated to providing a relatively fast, robust, and cost-effective method for the analysis of methylated cytosine at nucleotide resolution in different types of RNA.


5-methylcytosine (5mC) is one of the most common and abundant methylation modifications in the transcriptome, and is highly prevalent in mRNAs as well as in some non-coding RNAs. 5mC is usually located upstream of guanine in DNA double helix and is methylation of the fifth position of cytosine. 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. As one of the most-studied cytosine modifications and major epigenetic modifications, the detection and quantification of 5mC are essential for understanding gene expression and other processes subjected to epigenetic regulation. We provide comprehensive RNA BS-seq service that allows unbiased whole-transcriptome RNA methylation analysis. The technology uses sodium bisulfite to convert un-methylated cytosine into uracil, while methylated cytosine remains intact. After this step, PCR amplification, library preparation, and next-generation sequencing are performed. Finally, by comparing untreated and sodium bisulfite-treated sequences, it can determine which nucleotide positions are methylated. As a mainstream method for detecting methylated cytosine, RNA BS-seq can provide single-nucleotide resolution and full genome coverage, and is the gold standard method for methylation profile analysis. RNA BS-seq service can comprehensively analyze the distribution and changes of m5C in transcriptome, help researchers understand the role of RNA modification in life and diseases, and promote accurate diagnosis and treatment of diseases.


High Coverage Single-base Resolution High Precision One-stop Service
The method can comprehensively detects m5C sites of multiple RNA molecules. Provides single-base resolution with full genome coverage. High precision allows un-methylated C conversion efficiency over 99%. 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;
cDNA library preparation.


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

RNA Bisulfite Sequencing

In-depth data analysis:

  • Sequencing depth and coverage analysis
  • mC calling
  • Transcriptome-wide profiling of m5C methylation
  • Differential binding analysis
  • Methylation level and density analysis
  • Global trends of methylome
  • DMR annotation and enrichment analysis
  • Clustering analysis

Sample Requirements

RNA sample quantity ≥ 50 ug.
1.8 ≤ OD260/280 ≤ 2.0.
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.


  1. Zhao L Y, Song J, Liu Y, et al. Mapping the epigenetic modifications of DNA and RNA. Protein Cell, 2020, 10.
  2. Huang T, Chen W, Liu J, et al. Genome-wide identification of mRNA 5-methylcytosine in mammals. Nat Struct Mol Biol, 2019, 26(5): 380-388.
  3. Chen Y, Pal B, Visvader J E, et al. Differential methylation analysis of reduced representation bisulfite sequencing experiments using edgeR. F1000Res, 2017, 6: 2055.
* For Research Use Only. Not for use in diagnostic procedures.


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