Identification of genes related to stress and withanolide biosynthesis in roots of W. somnifera VA-1 - Control-1
The RNA samples with qualified RIN were taken for library preparation using TruSeq RNA Library Prep Kit (Illumina, Inc, USA). Starting with total RNA, mRNA was isolated from Total RNA through a technique known as polyA-selection, where polyadenine (polyA) tails present at the end of mRNA molecules are selectively captured using oligo-dT probes. After the polyA-selection, the isolated mRNA molecules are subjected to fragmentation which is followed by synthesis of first and second strand cDNA. End-repairing and A-tailing was done inorder to ligate the Illumina adapters. The prepared mRNA libraries were quantified using QuantStudio Real-Time PCR Systems (Thermo Fisher Scientific, USA) and Qubit 4.0 fluorometer (Invitrogen, Thermo Fisher Scientific, USA). The insert size of the library was assessed using 2100 Fragment Analyzer System (Agilent Technologies, USA). The library with appropriate insert size of 300 - 350 bp and effective concentration of more than 10 nM were pooled and paired end sequencing was performed on Illumina NovaSeq X platform (Illumina, Inc, USA), with the read length of 150 bp.