Single-Nucleotide RNA Pseudouridine (Ψ) Modification Seq (BID-seq)

01What the service reports

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: the base is left unconverted and becomes a deletion signature during reverse transcription, and that signature both locates and quantifies the site.

The readout is direct rather than captured. Nothing is enriched before sequencing, so the signal at a position comes from the conversion chemistry itself, and each call arrives with a modification ratio rather than only an enrichment score.

Arraystar runs BID-seq as a complete laboratory workflow: RNA sample QC, bisulfite treatment, library construction, sequencing and bioinformatics. Sites are annotated to the reference transcriptome with genomic coordinates, host gene and functional region, and a study can be scoped to mRNA alone or to mRNA together with long non-coding RNA.

02Benefits

• A signature at the base: the position is read out of the alignment itself, so each pseudouridine call is a single base rather than a peak.
• A level as well as a position: the conversion-based readout reports how much of a site is modified.
• Antibody-free: there is no immunoprecipitation, so the readout does not depend on antibody cross-reactivity or on how efficiently a pull-down performed.
• Annotated output: sites are placed on the reference transcriptome with coordinates, host gene and functional region attribution.
• Fitted to downstream questions: site-level data serves work on translation control, transcript stability, coding-region function and gene regulation.

03Background and detection principle

Pseudouridine shapes the structure of an mRNA and how readily that transcript is translated, so a map is most useful when it resolves individual positions. Region-level enrichment leaves both the position and the level of a site open, and it folds the preferences of the capture reagent into the signal.

BID-seq rests on a chemical difference instead. Pseudouridine resists bisulfite deamination, so it stays unconverted while unmodified positions react; when the treated RNA is reverse transcribed, the resistant base produces a deletion signature. Because that signature sits exactly at the modified position, the alignment places the site and the readout gives its level in the same pass.

The same workflow extends to long non-coding RNA, so coding and non-coding transcripts can be profiled together within one experiment.

Pseudouridine resists the bisulfite reaction, so the base survives and reverse transcription writes a deletion at that position — the site and its level come out of the same signature.Dai Q. et al., Nature Biotechnology 2023 · DOI 10.1038/s41587-022-01505-w

04Workflow

Five steps take a sample from RNA to single-base pseudouridine data:

1. RNA sample QC, covering integrity and purity before treatment.
2. Bisulfite treatment, which converts unmodified positions while pseudouridine resists.
3. Library construction from the treated RNA.
4. Sequencing of the BID-seq library.
5. Bioinformatics analysis to call the deletion signatures and quantify Ψ stoichiometry.

05Bioinformatics and deliverables

Reads are aligned to the reference transcriptome, bisulfite-induced deletion signatures are detected, and pseudouridine stoichiometry is quantified at single-nucleotide resolution. Motif characterization, transcript-feature distribution, differential modification, functional enrichment and genome-browser visualization are layered on top of the site calls.

Deliverables

• Raw pseudouridine mRNA sequencing data files (FASTQ), formatted for GEO/SRA submission.
• Sample and sequencing QC reports with sorted BAM alignment files and core mapping statistics.
• High-confidence single-base pseudouridine site tables (XLSX) listing coordinates, host genes and stoichiometry.
• Single-base pseudouridine transcript-feature distribution figures and consensus motif logos (PDF/PNG).
• Single-base differential pseudouridine tables (XLSX) with fold change and significance.
• GO enrichment reports for pseudouridine-modified genes (HTML/PDF/PNG).
• Genome-browser track files (bigWig/bedGraph) together with a structured project report.

06Research applications

• Transcriptome-wide maps: pseudouridine sites are resolved across protein-coding transcripts and, where the study is scoped that way, across lncRNAs as well.
• Translation and transcript stability: site-level data can be set against how efficiently an mRNA is translated and how long it persists.
• Coding-region and UTR function: sites are localized within CDS, UTR and splice-adjacent regions, so positional effects can be examined rather than assumed.
• Disease-oriented work: differentially modified mRNA sites can be carried forward as candidate biomarkers.
• Method cross-checks: because the readout is chemical rather than capture-based, the site list can be compared with results from other Ψ mapping routes without sharing their reagent bias.

07Sample requirements

Storage. Cells and tissues: preserve in TRIzol or an RNA stabilisation 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 repeated freeze–thaw cycles.

Shipping. Place the sample in a 1.5 mL RNase-free microcentrifuge tube, seal it with parafilm or a cap lock, and ship on dry ice with adequate insulation.

08FAQ

09References

1. Dai Q, Zhang LS, Sun HL, et al. 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