Eukaryotic mRNA Sequencing with Reference

mRNA sequencing is a prominent content in transcriptome sequencing, which can truly reflect the expression and regulation information of genes. CD Genomics can provide mRNA sequencing services for various eukaryotic and prokaryotic species. Among them, eukaryotic mRNA sequencing based on reference sequences can comprehensively obtain transcript information of species-specific cell, tissues, or organs, so as to conduct eukaryotic transcript structure analysis, variation analysis, gene expression level analysis, novel transcript discovery, and more.

Overview

The research object of eukaryotic mRNA sequencing is a collection of mRNAs transcribed from a specific cell or tissue in a certain functional state. This technology uses oligo-dT magnetic beads to capture RNA with polyA tails for library construction and sequencing. It can study mRNA differential expression or detect structural variation, and screen molecular markers associated with diseases or traits; it can also reveal the complexity of the transcriptome, determine gene and transcript structure, alternative splicing, and Novel Transcripts. Eukaryotic mRNA sequencing has been widely used in fundamental research, molecular breeding, clinical diagnosis, drug development and many other fields.

Eukaryotic mRNA sequencing can be achieved with high-throughput sequencing. CD Genomics's eukaryotic mRNA sequencing uses the Illumina sequencing platform to analyze all mRNAs transcribed in a specific eukaryotic tissue or cell in a specific state. Sequencing and comparison with the reference genome can not only comprehensively and rapidly analyze the mRNA sequence and abundance information, but also analyze the gene structure and the new transcripts generated.

Features

Good Fidelity & Reliability Experienced Scientist Team Fast and Efficient Professional Bioinformatics
Profiling gene expression more realistically; Transcripts of different abundances can be accurately quantified. Can provide a full set of professional services from experimental design, sample testing, data analysis, etc. Mature process reduces unnecessary waste of samples and time, and the turn-around time is short. Strong bioinformatic team provides conventional analysis and in-depth data analysis.

Project Workflow

Sample Preparation

1. Sample Preparation

RNA purification; quality assessment and quantification.

Library Preparation

2. Library Preparation

RNA fragmentation; crosslinking reaction; PCR amplification.

Sequencing

3. Sequencing Platform

Hiseq X Ten, PE150; 6 Gb per sample

Data Analysis

4. Data Analysis

Preprocess and visualize results, and perform custom bioinformatics analysis.

Eukaryotic mRNA Sequencing with Reference

Bioinformatics Analysis Pipeline

In-depth data analysis:

  • Sequencing data quality assessment (QC)
  • Reference sequence alignment
  • Statistical analysis of differential genes: cluster analysis; GO enrichment analysis; KEGG enrichment analysis
  • Gene structure analysis
  • New transcript prediction
  • Variable shear analysis
  • SNP and InDel analysis
  • Signal pathway network construction

Sample Requirements

Tissue sample: above 100 mg

Cell sample: cell amount: above 1×107

RNA sample: RNA quantity ≥ 5 μg; RNA purity: OD260/280 = 1.6~2.3; OD260/230 ≥ 1.5; RNA quality: 28S:18S ≥ 1.5 or RIN ≥ 7

Please make sure that the RNA is not significantly degraded.

Sample storage: Cell samples or fresh tissue pieces (cut into 5-10 mg pieces) can be treated with TRIzol or RNA protectant, frozen in liquid nitrogen, and stored at -80°C. RNA samples can be dissolved in ethanol or RNA-free ultrapure water, and stored at -80°C. Avoid repeated freezing and thawing during sample storage.

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. Bik HM, Porazinska DL, Creer S, et al. Sequencing our way towards understanding global eukaryotic biodiversity. Trends in ecology & evolution. 2012 Apr 1;27(4).
  2. Yassour M, Kaplan T, Fraser HB, et al. Ab initio construction of a eukaryotic transcriptome by massively parallel mRNA sequencing. Proceedings of the National Academy of Sciences. 2009 Mar 3;106(9).
* For Research Use Only. Not for use in diagnostic procedures.


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