Ribo-Seq (Ribosome Footprinting)

Ribo-Seq, also referred to as ribosome profiling or ribosome footprinting, is a next-generation sequencing (NGS)-based transcriptomic research technique that measures translation comprehensively and quantitatively. Ribo-Seq detected the precise positions of ribosomes to determine which mRNAs are being actively translated, which allows for the profiling of the active ribosomes in a cell at a particular moment, as well as the location of the translation initiation sites, the complement of translated ORFs, the specific locations of ribosomes on the mRNA, and the translation rate of ribosomes.

Overview

Compared to RNA-Seq, Ribo-Seq only targets mRNA sequences protected by the ribosome during the process of decoding by translation, which unveils the composition and regulation of the expressed proteome. With the effect of translation inhibitors, the ribosome being translated is fixed to the mRNA sequence or translation initiation site. After cell lysis, RNase was added into the cell lysate to digest the mRNA which is not protected by ribosomes. The single ribosome is isolated and the short fragments of mRNA which are not digested on the ribosome are extracted and purified for library building sequencing and corresponding data analysis. Ribosome profiling is readily adaptable to a diversity of organisms, which allows for the high-precision investigation of protein translation at the genomic level.

Features

High Throughput High Accuracy High Repeatability One-stop Service
Obtain more than 5 G information in one sequencing. Accurate reference library and high sequence coverage;
Proteins can be identified indirectly.
Deep sequencing ensures the randomness of detection and does not require technical repetition. Provides one-stop services for library construction, sequencing, sample QC and data analysis.

Project Workflow

Sample Preparation

1. Sample Preparation

RNA purification; quality assessment and quantification.

Library Preparation

2. Library Preparation

Size selection; strand-specific cDNA libraries.

Sequencing

3. Sequencing

Illumina HiSeq; PE 150.

Data Analysis

4. Data Analysis

Visualize and preprocess results, and perform custom bioinformatics analysis.

Bioinformatics Analysis Pipeline

Ribo-Seq (Ribosome Footprinting)

In-depth data analysis:

  • Data quality control;
  • Reference-based mapping;
  • Gene expression level evaluation;
  • Clustering;
  • GO and KEGG pathway analysis;
  • Differential gene analysis (translational level);
  • Translation ratio analysis;
  • Protein-protein interaction network analysis.

Sample Requirements

Ribosome protected fragment (RPF) samples: concentration ≥ 200 ng/ µL, amount ≥ 2 µg, volume ≥ 10 µL, main peak 25 – 38 nt.

Please make sure that the RNA is not degraded nor contaminated.

Tissue or cell samples: We can offer RNC (Ribosome Nascent - chain Complex) separation and mRNA extraction service.

Sample storage: RNA can be dissolved in RNA Stable 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 a 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. Wang H, Wang Y, Xie Z. Computational resources for ribosome profiling: from database to Web server and software. Briefings in Bioinformatics. 2017, 20.
  2. Popa A, Lebrigand K, et al. RiboProfiling: a Bioconductor package for standard Ribo-seq pipeline processing. F1000 Research. 2016, 5.
  3. Ingolia NT, Ghaemmaghami S, Newman JR, et al. Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science. 2009, 324(5924): 218-23.
* For Research Use Only. Not for use in diagnostic procedures.


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