RNA-Seq analysis and de novo transcriptome assembly of low temperature responsive transcripts of Hevea brasiliensis
Transcriptome library for sequencing was constructed according to the Illumina TruSeq RNA library protocol outlined in “TruSeq RNA Sample Preparation Guide”. Briefly, mRNA was purified from 1 microgram of intact total RNA using oligodT beads. The purified mRNA was fragmented for 4 minutes at elevated temperature (94°C) 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 Rnase H. 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 11 cycles of PCR for enrichment of adapter ligated fragments.