Pseudo Sequencing

Pseudouridylation sequencing is a next generation sequencing (NGS)-based method to comprehensively detect global maps of pseudouridylation, a post-transcriptional modification in various molecules. It can quickly identify the complete picture of the pseudo-uracil modification. Our experienced technical team provides you with integrated pseudo sequencing service and professional data analysis.

Overview

Pseudouridylation, often referred to as the fifth nucleotide and isomerized from uracil nucleoside (U) to pseudo-uracil (ψ), is one of the most abundant stable RNA internal post-translational modifications. It is ubiquitous in evolutionarily conserved and functionally important regions of rRNA, tRNA and other non-coding RNAs (ncRNAs), which can reduce RNA conformational variability, enhance base pairing stability and polar interactions with proteins. Pseudo-uracil modification not only contributes to RNA structure stability and ribosome biogenesis and activity, pre-mRNA splicing and protein synthesis, but also participates in regulation of growth, development and stress response of different organisms. We provide integrated pseudo sequencing, a technology combines high-throughput sequencing techniques with bioinformatics analysis to identify pseudo-uracil sites at single-nucleotide resolution. The method uses oligo-dT to screen mRNA, and prepares randomly fragmented mRNA for library preparation, high-throughput sequencing, and pseudo-uracil modification sites analysis. Our experienced technical team can obtain millions of sequence tags in single sample, discover and search rare methylated modified fragments in the transcriptome, and help customers further understand the role and function of this new epigenetic transcriptome marker.

Features

Cost-efficiency Digitized Signal Professional Team Flexibility
The method covers all methylated regions of entire transcriptome. Direct quantification and sequencing of methylated fragments without cross-reactivity and background noise. Provide various in-depth data analysis to satisfy customer needs. Provide one-stop service from library preparation, sequencing to data analysis.

Project Workflow

Sample Preparation

1. Sample Preparation

RNA purification;
quality assessment and quantification.

Library Preparation

2. Library Preparation

RNA fragmentation;
specific library preparation.

Sequencing

3. Sequencing

Illumina HiSeq;
PE 50/75/100/150.

Data Analysis

4. Data Analysis

Visualize and preprocess results, and perform custom bioinformatics analysis.

Pseudo Sequencing

Bioinformatics Analysis Pipeline

In-depth data analysis:

  • Signal distribution of pseudo-uridine
  • Pseudo-uracil modification visualizaiton
  • Motif search
  • Differential gene screening of enrichment sites
  • Evolutionary conservative analysis
  • KEGG pathway enrichment analysis
  • Gene ontology analysis

Sample Requirements

RNA sample quantity ≥ 300 ug.
Please make sure that the RNA is not significantly degraded.

Sample storage: RNA can be dissolved in ethanol or RNA-free ultra-pure water and stored at -80°C. RNA should avoid repeated freezing and thawing.

Shipping Method: When shipping RNA samples, the RNA sample is stored in a 1.5 mL Eppendorf tube, sealed with sealing film. Shipments are generally recommended to contain 5-10 pounds of dry ice per 24 hours.

Deliverable: FastQ, BAM, coverage summary, QC report, custom bioinformatics analysis.

References:

  1. Carlile T M, Rojas-Duran M F, Gilbert W V. Pseudo-Seq: Genome-Wide Detection of Pseudouridine Modifications in RNA. Methods Enzymol, 2015, 560: 219-245.
  2. Sun L, Xu Y, Bai S, et al. Transcriptome-wide analysis of pseudouridylation of mRNA and non-coding RNAs in Arabidopsis. J Exp Bot, 2019, 15, 70(19): 5089-5600.
  3. Carlile T M, Rojas-Duran M F, Zinshteyn B, et al. Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells. Nature, 2014, 6, 515(7525): 143-146.
* For Research Use Only. Not for use in diagnostic procedures.


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