Single-Nucleotide RNA Dihydrouridine (D) Modification Seq (CRACI-seq)

01What the service reports

Single-Nucleotide RNA Dihydrouridine (D) Modification Seq (CRACI-seq) is an antibody-free, base-resolution method. A chemical conversion changes how dihydrouridine is read during reverse transcription, so a D site produces a signature of its own while unmodified positions read normally, and every site is mapped and quantified.

Because the readout comes from a conversion signature rather than from capture, a call arrives with both a position and a modification ratio. Sites sit on the reference transcriptome with genomic coordinates, host gene and functional region attribution.

Arraystar runs CRACI-seq end to end: RNA sample QC, the conversion reaction, library construction, sequencing and bioinformatics. A study can be scoped to mRNA alone, or to mRNA together with long non-coding RNA, so coding and non-coding transcripts are profiled within one experiment rather than two.

02Benefits

• Single-base D calls: each site is read from its own conversion signature instead of being inferred from an enriched region.
• A ratio, not just a call: the conversion-based readout reports how much of a site is modified, not only that it is.
• No antibody, no capture bias: the reaction works chemically, so nothing depends on antibody cross-reactivity or on how efficiently a pull-down performed.
• Annotated output: sites are placed on the reference transcriptome with genomic coordinates, host gene and functional region.
• Built for functional follow-up: site-level data serves work on translation control, transcript stability, coding-region function and gene regulation.

03Background and detection principle

Dihydrouridine shapes the structure and the stability of the transcripts that carry it, so its distribution matters at the level of individual nucleotides. Profiling that depends on enriching modified fragments reports regions and leaves both the position and the level of a site unresolved.

CRACI-seq resolves D through chemistry. A conversion reaction marks dihydrouridine so that reverse transcription records it as a distinct signature while unmodified positions read normally. One converted library therefore carries the position and the level of each site together, and sites in coding regions and in untranslated regions can be compared on the same footing.

Chemistry writes dihydrouridine into the readout as a signature of its own while unmodified positions read normally — so position and level come out of the same converted library.Ju C-W. et al., Nature Communications 2025 · DOI 10.1038/s41467-025-63918-w

04Workflow

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

1. RNA sample QC, covering integrity and purity before conversion.
2. Chemical conversion of the RNA, which marks dihydrouridine and writes its signature.
3. Library construction from the converted RNA.
4. Sequencing of the CRACI-seq library.
5. Bioinformatics analysis to call D sites and quantify their stoichiometry.

05Bioinformatics and deliverables

Reads are aligned to the reference transcriptome, CRACI-seq conversion signatures are detected, and D stoichiometry is quantified at single-nucleotide resolution. Motif characterization, differential modification, functional enrichment and genome-browser visualization are layered on top of the site calls.

Deliverables

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

06Research applications

• Transcriptome-wide D maps: dihydrouridine sites are resolved across protein-coding transcripts and, where the study is scoped that way, across lncRNAs as well.
• Transcript structure and stability: site-level D data can be set against how a transcript folds and how long it persists.
• Translation and gene regulation: modified positions can be related to translation control and to gene-regulation phenotypes.
• Coding-region and UTR localisation: sites are placed 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.

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. Ju C-W, Li H, Jiang B, et al. Quantitative CRACI reveals transcriptome-wide distribution of RNA dihydrouridine at base resolution. Nature Communications 2025;16(1):8863. DOI: 10.1038/s41467-025-63918-w