RNA Sequencing
Introduction
This method is also called as Metatranscriptomic sequencing or Metatranscriptomics, goes one step ahead of shotgun metagenomics and whole genome sequencing by looking at the RNA sample instead of DNA. The transcriptome is the complete set of transcripts in a cell, and their quantity, for a specific developmental stage or physiological condition. Understanding the transcriptome is essential for interpreting the functional elements of the genome and revealing the molecular constituents of cells and tissues, and also for understanding development and disease. The key aims of transcriptomics are: to catalogue all species of transcript, including mRNAs, non-coding RNAs and small RNAs; to determine the transcriptional structure of genes, in terms of their start sites, 5′ and 3′ ends, splicing patterns and other post-transcriptional modifications; and to quantify the changing expression levels of each transcript during development and under different conditions
Applications
•Global view of gene activity
•Novel gene and splice variant discovery
•Chromosomal rearrangement analysis
•Whole mRNA transcript expression analysis
Data Analysis
Primary Analysis
•Assembly and Mapping statistics
•Gene Expression profile
•SNPs and InDels calling
Secondary Analysis
•Differentially expressed genes (DEGs)
•Gene Annotations and Ontology Analysis
•Customised Analysis
Library Type: Eukaryotic RNA-Seq（cDNA library)
RNA Integrity Number(Agilent 2100)
Amount ≥ 6.8 (Animal), with smooth base line
Amount ≥ 6.3 (Plant and Fungus), with smooth base line
Purity (NanoDrop）
OD 260/280 = 1.8 - 2.2;
OD 260/230 ≥ 1.8;
Total RNA
Amount ≥ 0.4 μg
Total RNA (Blood)
Amount ≥ 0.8 μg
Total RNA (Single Cell)
Amount ≥ 100 ng
Amplified cDNA (double-stranded)
Amount ≥ 100 ng
Fragments between 400bp and 5000bp with main peak at ~2000bp
Purity (NanoDrop）
OD 260/280 = 1.8 - 2.0;
OD 260/230 ≥ 1.8;
Eukaryotic RNA-Seq (strand specific library)
Total RNA
Amount ≥ 0.8 μg
Amount ≥ 6.8 (Animal), with smooth base line
Amount ≥ 6.3 (Plant and Fungus), with smooth base line
Purity (NanoDrop）
OD 260/280 = 1.8 - 2.2;
OD 260/230 ≥ 1.8;