RNC-mRNA Sequencing

RNC sequencing is a next-generation sequencing (NGS)-based method to comprehensively profile Ribosome Nascent-chain Complex (RNC). RNC constitutes the ribosome attached to the polypeptide that it is synthesizing and is one of the most important factors in the translation process. The ratio of RNC mRNA to total mRNA refers to the translation ratio (TR) or translation efficiency. TR is closely related to the phenotype of cells, and is also regulated by UTR region, miRNA, etc. Studying the TR alteration of genes among different samples is significant for elucidating the molecular mechanism of ncRNAs. Meanwhile, it also serves a powerful tool for analyzing the occurrence and development of diseases, pharmacological action, molecular mechanisms of bio-development, and more.

Overview

In the RNC-Seq protocol, RNCs are generally enriched and sequenced with full-length mRNAs bound to multiple ribosomes by sucrose density gradient hyper-centrifugation. More specifically, the molecular weight increases as the mRNA binding to the ribosome to form a complex. And the more ribosomes are binding with mRNAs, the larger the molecular weight of the complex will be. Typically, an mRNA template can bind multiple ribosomes simultaneously, resulting in the synthesis of multiple protein molecules in parallel, so the mRNA molecule being translated will form polysomes with multiple ribosomes, which usually suspend in a high-density sucrose solution layer where the ongoing translating mRNAs can be separ¬¬ated. The following step is similar to RNA-Seq.

Features

High Throughput Transcriptome-Wide Bioinformatics Analysis One-stop Service
Cost-effective transcriptional profiling solution for large sample-size assays. Profile all mRNAs, known and novel. Our integrated bioinformatics pipeline can be tailored to suit your project. Provides one-stop service 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

RNA fragmentation;
cDNA 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.

RNC-mRNA Sequencing

Bioinformatics Analysis Pipeline

In-depth data analysis:

  • Data quality control;
  • Reference-based mapping;
  • Gene fusion detection;
  • Differential expression analysis;
  • Pathway analysis;
  • SNV & InDel detection;
  • Splicing analysis;
  • Prediction of novel mRNAs.

Sample Requirements

RNA sample (concentration ≥ 1 ng/µL, quantity ≥ 5 µg)
1.8 ≤ OD260/280 ≤ 2.2, OD260/230 ≥ 2.0, RIN ≥ 6.5, 28S:18S ≥ 1.0.
Please make sure that the RNA is not degraded nor contaminated.

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, full statistical analysis & alignments, custom analysis reports on customer request.

References:

  1. Li W, Ward FR, McClure KF, et al. Structural basis for selective stalling of human ribosome nascent chain complexes by a drug-like molecule. Nature structural & molecular biology. 2019;26(6):501-9.
  2. Lian X, Guo J, Gu W, et al. Genome-wide and experimental resolution of relative translation elongation speed at individual gene level in human cells. PLoS genetics. 2016;12(2):e1005901.
  3. Cabrita LD, Cassaignau AM, Launay HM, et al. A structural ensemble of a ribosome–nascent chain complex during cotranslational protein folding. Nature structural & molecular biology. 2016;23(4):278-85.
  4. Neuhof A, Rolls MM, Jungnickel B, et al. Binding of signal recognition particle gives ribosome/nascent chain complexes a competitive advantage in endoplasmic reticulum membrane interaction. Molecular biology of the cell. 1998;9(1):103-15.
* For Research Use Only. Not for use in diagnostic procedures.


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