13-1 stress leaf sample
Total RNA was isolated from sample using Trizol (Invitrogen) method for RNA extraction. The quality of the total RNA was checked on 1% denatured Agarose gel and quantified using Qubit fluorometer. The libraries were prepared using Lexogen SENSE mRNA-Seq Library Prep Kit V2 and as per its described protocol. The SENSE protocol comprises a highly specific bead-based poly(A) selection step which removes almost all traces of rRNA, tRNA, and non-polyadenylated RNA. Library production was initiated by the random hybridization of starter/stopper heterodimers to the poly(A) RNA still bound to the magnetic beads. These starter/stopper heterodimers contain Illumina-compatible linker sequences. A single-tube reverse transcription and ligation reaction extends the starter to the next hybridized heterodimer, where the newly-synthesized cDNA insert is ligated to the stopper. As the insert size is determined by the distance between starter/stopper binding sites, RNA fragmentation is not required. Therefore, spurious second strand synthesis from the 5 ends of fragments is absent, providing the basis for the excellent strand-specificity of the SENSE protocol. Second strand synthesis is performed to release the library from the oligodT beads, and the library is purified using magnetic beads, rendering the protocol highly suitable for automation. In a subsequent PCR amplification the complete sequences required for cluster generation are introduced. After obtaining the Qubit concentration for the library and the mean peak size from Bioanalyser profile, library was loaded into Illumina platform for cluster generation and sequencing.