RNA Bisulfite Sequencing Service

Through precise chemical conversion, RNA bisulfite sequencing enables single-nucleotide resolution mapping of 5-methylcytosine (m5C) across the transcriptome. CD BioSciences offers comprehensive, end-to-end RNA bisulfite sequencing services, enabling researchers to accurately profile m5C landscapes and investigate their functional roles in RNA stability, translation, and cellular regulation.

Introduction to RNA Bisulfite Sequencing

RNA bisulfite sequencing is a gold-standard, chemical-based technique for the genome-wide identification of 5-methylcytosine (m5C) at single-base resolution. The method leverages the differential reactivity of cytosine nucleotides: upon bisulfite treatment, unmethylated cytosines are deaminated to uracil (which are read as thymine during sequencing), whereas methylated cytosines (m5C) remain protected and unchanged. By comparing bisulfite-treated sequences to an untreated reference, every cytosine in the transcriptome can be interrogated for methylation status. This approach provides an unbiased, quantitative, and high-resolution map of m5C, offering critical insights into its distribution across mRNA, tRNA, rRNA, and non-coding RNA, and its implications in development, disease, and epigenetic regulation.

Fig.1 Optimization and validation of ultrafast bisulfite conditions using human 28S rRNA and tRNA. (Dai Q, et al., 2024)

Features of RNA Bisulfite Sequencing

RNA bisulfite sequencing is a robust and unbiased method that provides unparalleled accuracy and resolution for m5C detection, making it ideal for discovery and validation studies.

1. Single-Base Resolution: Precisely identifies the location of every m5C site across the transcriptome, enabling exact genomic coordinate mapping.
2. Unbiased Detection: Does not rely on antibodies, avoiding issues related to specificity, affinity, or epitope accessibility.
3. Quantitative Capability: Provides methylation levels at each site, allowing for comparative analysis across conditions or samples.
4. Broad RNA Compatibility: Applicable to all RNA types, including low-abundance transcripts, without prior enrichment.

Our Services

As a professional provider of epigenetic research services, CD BioSciences offers reliable, highly sensitive RNA bisulfite sequencing services that can comprehensively decode the m5C epitranscriptome in a sample. Using optimized bisulfite conversion protocols and advanced bioinformatics pipelines, we ensure accurate detection and quantification of methylation sites even from challenging or low-input material. Our service equips researchers with the precise data needed to explore the regulatory impact of m5C in processes such as stem cell differentiation, stress response, tumor progression, and neural function.

Workflow of RNA Bisulfite Sequencing Service

The RNA bisulfite sequencing workflow is an integrated, multi-stage process engineered to preserve RNA integrity during chemical conversion while maximizing sequencing accuracy and methylation call precision. Our pipeline begins with stringent sample QC and proceeds through optimized bisulfite conversion, strand-specific library construction, high-depth sequencing, and a specialized bioinformatics analysis pipeline designed to handle the unique complexities of bisulfite-converted RNA data.

RNA Integrity Assessment & Input Standardization

Total RNA undergoes rigorous quality control via Bioanalyzer or TapeStation analysis to ensure high integrity (RIN > 8.0). Input amounts are standardized based on sample type and research goals.

Bisulfite Conversion & Cleanup

RNA is treated with a high-efficiency bisulfite reagent under precisely controlled thermal cycling conditions to achieve >99% conversion of unmodified cytosines. The converted RNA is then purified using a column-based or bead-based cleanup protocol to remove salts and reagents.

Strand-Specific Library Preparation

Converted RNA is reverse transcribed using random primers and strand-specific adapters are ligated. The resulting cDNA is amplified with a limited number of PCR cycles to construct sequencing libraries while minimizing amplification bias.

High-Throughput Sequencing & QC

Libraries are quantified, pooled, and sequenced on Illumina platforms with 150bp paired-end reads to achieve sufficient depth (typically 50-100 million reads per sample) for accurate methylation calling across the transcriptome.

Specialized Bioinformatics Analysis

Sequencing data is processed through our dedicated pipeline including: adapter trimming, alignment to a bisulfite-converted reference genome using tools like Bismark or BS-Seeker2, methylation extraction at single-cytosine resolution, and filtering to remove potential false positives from incomplete conversion or sequencing errors.

Comprehensive Data Delivery & Biological Interpretation

Clients receive all raw data, methylation call files, summary statistics, visualization tracks for genome browsers, and a detailed report including differential methylation analysis, motif identification, and functional pathway enrichment based on annotated m5C sites.

Supported Sample Types

Our Advantages

1. Expertise in RNA Chemical Conversion: Optimized protocols maximize bisulfite conversion efficiency while preserving RNA integrity, ensuring high signal-to-noise ratios even with challenging samples.
2. Dedicated Bioinformatics Pipeline: Specialized analysis tools accurately distinguish true methylation signals from conversion artifacts and sequencing noise, delivering reliable single-base resolution data.
3. Proven Success Across Sample Types: Validated performance with diverse materials, from fresh tissues to low-input clinical specimens, ensures consistent and reproducible results.
4. Comprehensive Data Interpretation Support: Beyond standard reporting, in-depth analysis includes differential methylation profiling, motif discovery, and functional annotation to accelerate biological insights.

Accurate, base-resolution mapping of m5C is essential for understanding its role in RNA biology and disease. CD BioSciences' RNA bisulfite sequencing service provides the precision and depth required for these investigations. For a complete epitranscriptomic analysis, we also offer complementary services such as m5C RIP-seq for antibody-based enrichment studies and LC-MS/MS for global quantification of methylation levels, enabling fully customized and integrated research strategies. Contact us today to discuss your project and receive a detailed proposal.

Reference

1. Dai Q, Ye C, Irkliyenko I, et al. Ultrafast bisulfite sequencing detection of 5-methylcytosine in DNA and RNA[J]. Nature biotechnology, 2024, 42(10): 1559-1570.