title: Characterization of Labeo rohita Transcriptome: Sequencing, De Novo Assembly, Annotation and Mapping of genes associated with digestive and reproductive tissues.
design: mRNA was purified from one microgram of intact total RNA using oligo dT beads (TruSeq RNA Sample Preparation Kit, Illumina). The purified mRNA was fragmented at elevated temperature in the presence of divalent cations and reverse transcribed with Superscript II Reverse transcriptase by priming with Random Hexamers. Second strand cDNA was synthesized in the presence of DNA polymerase I and RnaseH. The cDNA was cleaned up using Agencourt Ampure XP SPRI beads (Beckman Coulter). Illumina Adapters were ligated to the cDNA molecules after end repair and addition of A base. SPRI cleanup was performed after ligation. The library was amplified using PCR for enrichment of adapter ligated fragments. The prepared library was quantified using Nanodrop and validated for quality by running an aliquot on High Sensitivity Bioanalyzer Chip (Agilent).