transcript expression evidence RNA-sequencing evidence RNAseq evidence whole transcriptome shotgun sequencing eco ECO:0000295 differential gene expression evidence from RNA-seq experiment RNA sequencing assay evidence RNA-seq evidence 2011-01-11T12:54:50Z mchibucos RNA sequencing ECO:0001821 WTSS ECO:0000357 Total RNA is isolated from cells and mRNA is purified by targeting the 3' polyadenylated (poly(A)) tail with poly(T) oligos covalently attached to a substrate. Next, cDNA fragments are generated either by hydrolysis of RNA into 200-300 base oligonucleotides, preceded by reverse transcription, or by reverse transcription initiated by random primers. The cDNA second strand is synthesized, fragments are adapter ligated and high-throughput sequencing is performed by Roche 454, Illumina, ABI-SOLiD, or another HT sequencing technology. Resulting sort reads are aligned against a reference genome, and downstream analysis is performed. A type of transcript evidence based on high-throughput (HT) sequencing of fragmented cDNA molecules.