Strand-Specific RNA Sequencing

Ordinary RNA sequencing libraries cannot retain the orientation information of transcripts, but many gene regions have transcripts of both positive and negative strands, especially most non-coding RNAs come from antisense strands. Strand-specific RNA sequencing is a next-generation sequencing technology that allows for the orientation information of RNA strands which are stored in the sequencing library during the library construction process.  The data analysis after sequencing can determine whether the transcript comes from the sense or antisense strand.

Overview

The non-strand-specific transcriptome sequencing information does not contain the strand orientation information, and cannot accurately judge the sense and antisense transcripts, resulting in the failure to reflect the real state of transcription. And strand-specific RNA seq can solve this problem. Strand-specific library building is done by replacing dTTP with dUTP when synthesizing the second strand of the cDNA. After the strand is synthesized, the second strand of cDNA is degraded with USER enzyme and PCR amplification is performed to complete the preparation of the library. This method can count the number of transcripts and determine the structure of genes more accurately, and can find more antisense transcripts. It is currently widely used in the study of gene structure and gene expression regulation.

CD Genomics provides one-stop strand-specific transcriptome sequencing services, covering sample preparation, library construction, sequencing, and data analysis. Strand-specific library building methods are slightly different depending on prokaryotes or eukaryotes. The strand-specific library construction can be used for transcriptome sequencing of species with and without reference. Our strand-specific sequencing services can obtain more accurate gene expression quantification, improve the accuracy of circRNA discovery, identify non-coding antisense transcripts, better predict new transcripts, and more accurately analyze and identify operons.

Features

Good Fidelity & Reliability High Data Richness Experienced Scientist Team Professional Bioinformatics
Profiling gene expression with improved accuracy; allows for the orientation information of RNA strands. Antisense transcripts can be found accurately and more relevant information can be obtained for identification and analysis. Can provide a full set of professional services from experimental design, sample testing, data analysis, etc. 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

Illumina HiSeq; /100/150
Eukaryotes: 5-10G
Prokaryotes: 1-10G

Data Analysis

4. Data Analysis

Preprocess and visualize results, and perform custom bioinformatics analysis.

Strand-Specific RNA Sequencing

Bioinformatics Analysis Pipeline

In-depth data analysis:

  • Sequencing data quality control
  • Gene expression analysis
  • Alternative splicing analysis (eukaryotes)
  • Gene structure optimization and new transcript prediction
  • SNP analysis, InDel analysis, SSR analysis, and ORF prediction
  • Chain orientation analysis
  • Gene differential expression analysis
  • KEGG and GO functional analysis of differentially expressed genes

Sample Requirements

Tissue sample: plant sample > 500mg; animal sample > 300mg

Cell sample: cell amount: above 5×106

Blood sample: Whole blood/whole blood isolated nucleated cells >3mL

RNA sample: RNA quantity: 30-300 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: 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: The samples should be 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. Levin JZ, Yassour M, Adiconis X, et al. Comprehensive comparative analysis of strand-specific RNA sequencing methods. Nature methods. 2010 Sep;7(9).
  2. Yassour M, Pfiffner J, Levin JZ, et al. Strand-specific RNA sequencing reveals extensive regulated long antisense transcripts that are conserved across yeast species. Genome biology. 2010 Aug;11(8).
* For Research Use Only. Not for use in diagnostic procedures.


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