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Single-Base RNA Modification Sequencing
Single-Nucleotide RNA Pseudouridine (Ψ) Modification Seq(BID-seq)
Single-Nucleotide RNA Pseudouridine (Ψ) Modification Seq(BID-seq) is an antibody-free, base-resolution method. Bisulfite-induced deletion sequencing exploits the resistance of pseudouridine to bisulfite to create a deletion signature, mapping and quantifying Ψ at single-base resolution.
Arraystar Single-Nucleotide RNA Pseudouridine (Ψ) Modification Seq(BID-seq) is an end-to-end sample-to-data service for mRNA, from RNA sample QC and mRNA treatment through library construction, sequencing, and bioinformatics. Sites are annotated to the reference transcriptome with genomic coordinates, host gene, and functional region attribution.
Benefits
In mRNA, pseudouridine influences structure and translation, so base-resolution mapping is informative. Bisulfite-induced deletion sequencing exploits Ψ resistance to create a deletion signature, mapping and quantifying each site at base resolution.
Single-base resolution: pinpoints Ψ sites in mRNA.
Quantitative stoichiometry: conversion-based readouts give accurate modification ratios.
Antibody-free and unbiased: avoids enrichment cross-reactivity and pull-down bias.
Functional annotation: Sites are annotated to the reference transcriptome with genomic coordinates, host gene, and functional region attribution.
Application-ready: supports translation control, RNA stability, coding-region function, and gene-regulation studies across the mRNA transcriptome.
| Service Name | RNA Class | Price |
|---|---|---|
| Single-Nucleotide RNA Pseudouridine (Ψ) Modification Seq(BID-seq) | mRNA | |
| Single-Nucleotide RNA Pseudouridine (Ψ) Modification Seq(BID-seq) | mRNA & lncRNA |
Background
In mRNA, the modification shapes transcript stability, translation, and gene regulation, so base-resolution mapping across coding and untranslated regions is what distinguishes it. Bisulfite-induced deletion sequencing exploits the resistance of pseudouridine to bisulfite deamination, so reverse transcription creates a deletion signature at each Ψ site while unmodified positions read normally. This antibody-free readout maps each Ψ site at single-base resolution. The same workflow extends to long non-coding RNAs (lncRNAs), enabling researchers to profile this modification on lncRNA transcripts in parallel with mRNA in a single assay.
The method was validated for mRNA, sites are annotated to the reference transcriptome with genomic coordinates, host gene, and functional region attribution. (Dai Q, et al. He C. Nature Biotechnology 2023;41(3):344-354., DOI: 10.1038/s41587-022-01505-w).
Key concept: Single-Nucleotide RNA Pseudouridine (Ψ) Modification Seq(BID-seq) is a base-resolution method in which bisulfite-induced deletion sequencing exploits the resistance of pseudouridine to bisulfite to create a deletion signature, mapping and quantifying Ψ at single-base resolution.
Figure 1. BID-seq pseudouridine detection scheme. Bisulfite chemistry triggers a deletion signature at each pseudouridine site, while unmodified positions read normally, mapping and quantifying Ψ at single-base resolution.
Workflow
Five steps from RNA sample to single-base pseudouridine (Ψ) data:
Figure 2. Single-Nucleotide RNA Pseudouridine (Ψ) Modification Seq workflow.
Bioinformatics
The bioinformatics pipeline aligns reads to the reference transcriptome, detects bisulfite-induced deletion signatures, and quantifies Ψ stoichiometry at single-nucleotide resolution, with motif, distribution, differential, enrichment, and genome-browser analyses.
Deliverables
- Raw Ψ mRNA sequencing FASTQ data files, formatted for GEO/SRA public database submission.
- Sample and sequencing QC reports with sorted BAM alignment files and core mapping statistics.
- High-confidence single-base Ψ site annotation tables (XLSX) with coordinates, genes, and absolute stoichiometry.
- Single-base Ψ transcript feature distribution figures and consensus Ψ motif logos (PDF/PNG format).
- Single-base resolution differential Ψ methylation tables (XLSX) with fold change and significance metrics.
- Gene Ontology (GO) enrichment analysis reports for Ψ-modified genes, provided in HTML/PDF/PNG formats.
- Genome browser-compatible track files (bigWig/bedGraph) and a full structured Ψ analysis project report.
Research Applications
- Transcriptome-wide mapping of the modification across protein-coding and non-coding mRNA.
- Translational control: relates base-resolution modification to mRNA translation and stability.
- Coding-region function: localizes sites within CDS, UTR, and splice-adjacent regions.
- Disease-oriented studies: finds differentially modified mRNA sites as biomarkers.
References
[1] Dai Q, Zhang LS, Sun HL, et al. He C. Quantitative sequencing using BID-seq uncovers abundant pseudouridines in mammalian mRNA at base resolution. Nature Biotechnology 2023;41(3):344-354. DOI: 10.1038/s41587-022-01505-w.
Sample Requirements
Storage Guidelines
Cells and Tissues: Preserve in TRIzol or an RNA stabilization solution; snap freeze in liquid nitrogen and store at –80 °C.
RNA: Resuspend in ethanol or RNase-free ultrapure water; store at –80 °C and avoid multiple freeze-thaw cycles.
Shipping Instructions
Place the sample in a 1.5 mL RNase-free microcentrifuge tube.
Seal the tube with parafilm or a cap lock to ensure sample integrity.
Ship the package on dry ice with adequate insulation to maintain the required temperature.
| Sample Type | Notes |
|---|---|
| Whole blood | Use EDTA tubes only, as heparin is not compatible with subsequent analytical procedures. |
| Cultured cells | Submission of cell pellets is preferred to ensure high-quality material for processing. |
| Tissue | Provide fresh or frozen specimens, ensuring that necrotic material is strictly avoided. |
| Total RNA | Maintain an OD 260/280 ratio ≥ 1.8 and RIN ≥ 7 with no visible degradation. Submit at least 10 µg total RNA. |
FAQ
Which mRNA features are annotated?
Sites are annotated to the reference transcriptome with genomic coordinates, host gene, and functional region attribution. This lets you interpret each site in the context of its host gene and mRNA functional region. The method supports both mRNA and tRNA workflows, so you can choose the readout that matches your study.
Can this method quantify the modification at single-base resolution?
Yes. Bisulfite-induced deletion sequencing exploits the resistance of pseudouridine to bisulfite deamination, so reverse transcription creates a deletion signature at each Ψ site while unmodified positions read normally. This provides single-base resolution and absolute stoichiometry for each site in mRNA.
How does this method differ from antibody-based mRNA profiling?
Antibody-based methods rely on enrichment and offer limited resolution with intrinsic motif bias. This method is antibody-free and delivers base-resolution, quantitative calling in mRNA, avoiding cross-reactivity and enrichment bias. This suits projects needing quantitative site-level mapping rather than region-level enrichment, and it pairs well with orthogonal validation.
How does this service support pseudouridine research?
By mapping Ψ at single-base resolution, the service helps researchers explore its roles in RNA structure and function, strengthening the evidence for impactful studies.
Why is high-resolution pseudouridine detection valuable?
Accurate Ψ mapping provides robust site-level evidence, making it a valued asset for advancing RNA biology.
Does this service also profile lncRNA?
Yes. We provide two service options: mRNA modification, and mRNA plus long non-coding RNA (lncRNA) modification. Both transcript types can be analyzed together in a single experiment, so there is no need to split them into two projects, and the scope of your study opens up accordingly.
!This assay is provided for research applications only and is not intended for clinical diagnosis, treatment, or personal health evaluation.