Analysis of leaf and root transcriptome of Bacopa monnieri
Library preparation was performed at Genotypic Technology’s Genomics facility following SureSelect Strand-Specific RNA Library Prep Kit protocol outlined in “SureSelect Strand-Specific RNA Library Prep for Illumina Multiplexed Sequencing mRNA Library Preparation Protocol” (Cat # 5500-0116). Briefly, the mRNA was fragmented for 4 minutes at elevated temperature (94oC) in the presence of divalent cations and First strand cDNA is synthesized. Strand Specificity is maintained by the addition of Actinomycin D at this step. The single stranded cDNA was cleaned up using AgencourtAmpure XP SPRI beads (Beckman Coulter). Second strand cDNA was synthesized using second strand synthesis mix. The cDNA was cleaned up using AgencourtAmpure XP SPRI beads (Beckman Coulter). Adapters were ligated to the cDNA molecules after end repair and addition of “A”- base. SPRI cleanup was performed after ligation. The library was indexed and amplified using 10cycles of PCR for enrichment of adapter ligated fragments. The prepared library was quantified using Nanodrop and qubit and validated for quality by running an aliquot on High Sensitivity Bioanalyzer Chip (Agilent). (Refer Figure 1 for Bioanalyzer profiles of amplified product ePCR1). Sequencing will be followed on NextSeq 500 sequencer (Illumina) at Genotypic Technology’s Genomics facility