Cultivated  annual Nagouri ecotype Ashwagandha
30ug of qubit quantified total RNA was taken and enriched for PolyA using MicroPolyA Purist. The enrichment was assessed by running an aliquot on RNA Nano Bioanalyzer chip. Transcriptome library for sequencing was constructed according to the NEXTflex Directional RNA-Seq (dUTP based)library protocol outlined in NEXTflex Directional RNA-Seq (dUTP based)Sample Preparation Guide (Cat # 5129-050). Briefly, the rRNA depleted RNA was fragmented for 10 minutes at elevated temperature (95oC) in the presence of divalent cations and reverse transcribed with Superscript II Reverse transcriptase by priming with Random Hexamers.The RNA-DNA hybrid was cleaned up using Agencourt Ampure XP SPRI beads (Beckman Coulter). Second strand cDNA was synthesized using second strand synthesis mix. Directionality is retained by the addition of dUTP at this step. The cDNA was cleaned up using Agencourt Ampure XP SPRI beads (Beckman Coulter). NEXTflex RNA-Seq Barcode 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 15 cycles 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)