Transcriptome of salt- tolerant and salt- susceptible pigeon pea genotypes under control and salt treated root tissue
Library preparation wasperformed at Genotypic Technology’s Genomics facility following Illumina TruSeq RNA library protocol outlined in “TruSeq RNA Sample Preparation Guide” (Part # 15008136; Rev. A; Nov 2010). 1ug of Total RNA was subjected to Poly A purification of mRNA. Purified mRNA wasfragmented for 2minutes at elevated temperature (94oC) in the presence of divalent cations and reverse transcribed with Superscript IIIReverse transcriptase inthe presence of First strand mix. Second strand cDNA was synthesized using Second strand mix. 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 8cycles of 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). (Refer Figure 1 for Bioanalyzer profiles of amplified product ePCR1)