tRNA Pseudouridine BID-Seq | Base-Resolution Ψ Mapping on tRNA

Overview

Pseudouridine detected through a deletion signature

Pseudouridine is the most abundant modified base in RNA, and in tRNA it stabilises structure and supports decoding. Because Ψ is an isomer of uridine rather than an added chemical group, it cannot be detected by looking for a novel adduct; the assay has to exploit a difference in chemical behaviour instead.

Arraystar Single-Nucleotide tRNA Pseudouridine (Ψ) Modification Seq(BID-seq) is an end-to-end sample-to-data service for tRNA, from RNA sample QC and tRNA treatment through library construction, sequencing and bioinformatics. Sites are localized within mature tRNA transcripts with relative coordinates, gene attribution, anticodon and isotype, and structural-domain context.

What is tRNA BID-seq?

BID-seq is an antibody-free, base-resolution method for pseudouridine on tRNA. Bisulfite treatment leaves Ψ resistant while other bases react, so reverse transcription produces a deletion signature at each modified position and Ψ is mapped and quantified at single-base resolution.

Service at a Glance

Standard tRNA pseudouridine project — custom designs and add-on analyses available on request

Service Name	Price
Single-Nucleotide tRNA Pseudouridine (Ψ) Modification Seq(BID-seq)

Benefits

What BID-seq gives you on tRNA

Deletion signature at the modified base

The read-out marks each Ψ position through a defined deletion event rather than through enrichment breadth.

Stoichiometry per site

The frequency of the signature at a position yields the modification fraction for quantitative comparison.

Antibody-free detection

Chemical detection avoids pull-down bias and cross-reactivity inside tightly folded tRNA.

Mature-tRNA annotation

Sites carry relative coordinates, host tRNA gene, anticodon, isotype and structural-domain assignment.

TΨC-focused questions

Domain assignment lets the TΨC arm and other domains be examined separately.

Single-project delivery

RNA QC, tRNA treatment, library construction, sequencing and analysis arrive together.

Background

Why pseudouridine needs a chemistry-based read-out

Pseudouridine changes the way the base links to the ribose, which alters backbone flexibility and base stacking rather than the base-pairing surface alone. In tRNA that translates into structural stability and support for decoding, and the effect depends on which position carries the modification (1).

Bisulfite-induced deletion sequencing exploits the resistance of pseudouridine to bisulfite deamination: reverse transcription creates a deletion signature at each Ψ site while unmodified positions read normally. Arraystar runs the chemistry in a tRNA workflow and reports each site with its tRNA context (1).

tRNA Pseudouridine BID-Seq Workflow

From total RNA to base-resolution Ψ calls

The workflow runs sample QC with small-RNA-retaining purification, tRNA processing and bisulfite treatment, library construction and sequencing, then deletion-aware alignment that converts the signature into per-site Ψ calls.

tRNA Pseudouridine BID-Seq Workflow — Figure 2. Single-Nucleotide tRNA Pseudouridine (Ψ) Modification Seq workflow.

Bioinformatics & Deliverables

Bioinformatics for BID-seq

Reads are aligned to a curated tRNA reference, deletion-type signatures are called as pseudouridine positions, and per-position stoichiometry is reported with motif, distribution, differential, enrichment and genome-browser outputs.

Standard Deliverables

Raw sequencing reads (FASTQ) formatted for GEO/SRA deposition.
QC report with sorted BAM files and core mapping statistics.
Single-base Ψ site tables (XLSX) with coordinates, anticodon, isotype and stoichiometry.
Ψ distribution and motif figures across tRNA domains (PDF/PNG).
Differential Ψ tables (XLSX) with fold change and significance.
Genome-browser tracks (bigWig/bedGraph) with a structured tRNA report.

Research Applications

Research directions for tRNA pseudouridine

Structural stabilisation

Relates Ψ at a defined position to tRNA folding and stability.

TΨC arm function

Uses domain assignment to examine the TΨC arm separately from other regions.

Decoding and translation

Places Ψ in anticodon context to test its effect on decoding.

Isotype-resolved comparison

Contrasts Ψ occupancy between tRNA isotypes and isoacceptors.

Disease-oriented screening

Identifies differentially pseudouridylated positions between groups.

Sample Requirements

Sample handling and submission for tRNA pseudouridine

Sample Storage

For cells/tissue: use TRIzol or an RNA-stabilizing reagent, quick-freeze in liquid nitrogen, and keep at –80 °C.
For RNA: dissolve in ethanol or RNase-free water, store at –80 °C, and limit freeze–thaw cycles.

Shipping Instructions

Transfer each sample into a 1.5 mL nuclease-free tube.
Close the tube securely with parafilm or a cap lock to preserve integrity.
Send on dry ice with sufficient insulation to sustain the required temperature.

Sample Type	Notes
Whole blood	Use EDTA tubes only; heparin is not compatible with subsequent analytical procedures.
Cultured cells	Cell pellets are preferred to ensure high-quality material for processing.
Tissue	Provide fresh or frozen specimens and avoid necrotic material.
Total RNA	Maintain an OD 260/280 ratio ≥ 1.8 and RIN ≥ 7 with no visible degradation. Submit at least 10 µg total RNA.

Submit at least 10 µg total RNA per sample; the amount includes material consumed by sample QC. Questions: support@arraystar.com

FAQ

Questions we are asked about tRNA pseudouridine mapping

Why does pseudouridine appear as a deletion rather than a substitution?

Ψ resists bisulfite deamination, so the base is not converted the way cytidine is. When reverse transcriptase encounters the modified position it records a deletion event, and that signal marks the site at single-base resolution instead of indicating a modified region.

How is the Ψ fraction calculated?

The proportion of reads carrying the deletion signature at a given position reports the modification fraction, which is what makes the measurement quantitative rather than a simple presence call. Fractions at the same position can then be compared between samples and conditions.

Is the method antibody-dependent?

No. Detection is chemical: bisulfite treatment and the resulting deletion signature identify the site, so calling does not depend on antibody affinity or on pull-down efficiency in compact tRNA, and stoichiometry stays comparable between batches no matter how structured the target is.

Which tRNA features come with a site?

Each call is reported with relative coordinates inside the mature tRNA transcript, host tRNA gene attribution, anticodon and isotype, and structural-domain localization, so a Ψ position can be read within the tRNA that carries it rather than as a coordinate alone.

How much total RNA is needed?

At least 10 µg of total RNA per sample, including the material consumed by sample QC. Material should show an OD260/280 ratio of 1.8 or higher and no visible degradation; twice the minimum is recommended to avoid project delays. Contact support to discuss a lower amount.

How does BID-seq differ from protection-based Ψ methods?

BID-seq reads a deletion signature created when reverse transcription encounters Ψ, whereas protection-based chemistry shields the base so it appears as a retained signal. The two routes reach the same modification through different read-outs and can be used as independent confirmation.

Selected Publications

Key references for tRNA pseudouridine

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. PMID: 36302989

BID-Seq — Single-Nucleotide tRNA Pseudouridine (Ψ) Modification Seq