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Single-Base RNA Modification Sequencing
Single-Nucleotide RNA Pseudouridine (Ψ) Modification Seq(BACS-seq)
Single-Nucleotide RNA Pseudouridine (Ψ) Modification Seq(BACS-seq) is an antibody-free, base-resolution method. Controlled bisulfite chemistry protects pseudouridine and reads it as a distinct signature, while unmodified bases are converted, enabling absolute base-resolution Ψ quantification.
Arraystar Single-Nucleotide RNA Pseudouridine (Ψ) Modification Seq(BACS-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 shapes transcript structure and translation, so base-resolution mapping across coding regions is valuable. Controlled bisulfite chemistry protects Ψ and reads it as a distinct signature while other bases convert, mapping and quantifying each Ψ 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(BACS-seq) | mRNA | |
| Single-Nucleotide RNA Pseudouridine (Ψ) Modification Seq(BACS-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. Controlled bisulfite chemistry protects pseudouridine and reads it as a distinct signature, while unmodified positions are converted and read normally, giving absolute base-resolution pseudouridine quantification. 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. (Xu H, et al. Song CX. Nature Methods 2024;21(11):2024-2033., DOI: 10.1038/s41592-024-02439-8).
Key concept: Single-Nucleotide RNA Pseudouridine (Ψ) Modification Seq(BACS-seq) is a base-resolution method in which controlled bisulfite chemistry protects pseudouridine and reads it as a distinct signature while unmodified bases are converted, enabling absolute base-resolution Ψ quantification.
Figure 1. BACS-seq pseudouridine detection scheme. Controlled bisulfite chemistry protects pseudouridine and records it as a distinct signature, while unmodified bases are converted, enabling absolute base-resolution Ψ quantification.
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 maps reads to the reference transcriptome, recognizes BACS-seq conversion signatures, and quantifies pseudouridine 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] Xu H, Kong L, Cheng J, et al. Song CX. Absolute quantitative and base-resolution sequencing reveals comprehensive landscape of pseudouridine across the human transcriptome. Nature Methods 2024;21(11):2024-2033. DOI: 10.1038/s41592-024-02439-8.
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. Controlled bisulfite chemistry protects pseudouridine and reads it as a distinct signature, while unmodified positions are converted and read normally, giving absolute base-resolution pseudouridine quantification. 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 detecting Ψ with high accuracy, the service helps researchers study its functional significance, offering valuable insight for translational studies.
Why is accurate pseudouridine detection a valuable research asset?
Reliable, quantitative pseudouridine profiling strengthens the evidence base for research findings, making it a dependable tool for advancing the understanding of RNA function.
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.