Transcriptome analysis of Zingiber officinale
mRNA enrichment was done using NEBNext Poly (A) mRNA magnetic isolation module (Catalog: E7490, New England Biolabs). The enriched mRNAs were primed with NEBNext Random Primers and chemically fragmented in a magnesium-based buffer at 94°C for 10 minutes I order to get an insert of ~200 nucleotides. The fragmented mRNAs were reverse transcribed to form cDNA and the first strand cDNA reactions were converted to dS DNA. The double stranded cDNA fragments obtained were cleaned up by using 1.8X of AMPure XP beads (Catalog: A63881, Beckman Coulter). The DNA undergo end repair where in the mix converts the overhangs resulting from fragmentation into blunt ends. The 3' to 5' exonuclease activity of end repair mix removes the 3' overhangs and polymerase activity fills in the 5' overhangs. To the blunt ended fragments adenylation was performed by adding single 'A' nucleotide to the 3' ends. To the adenylated fragments loop adapters were ligated and cleaved with uracil-specific excision reagent (USER) enzyme. The samples were further purified using 0.9X AMPure XP beads (Catalog: A63881, Beckman Coulter) and the DNA was then amplified by 12 cycles of PCR using NEBNext Ultra II Q5 master mix, and to facilitate multiplexing, “NEBNext® Multiplex Oligos for Illumina” kit was used . The amplified products were purified using 0.9X AMPure XP beads (Catalog: A63881, Beckman Coulter) and the final DNA library was eluted in 15 uLs of 0.1X TE buffer. The RNA libraries were denatured using freshly prepared NaoH and applied onto Illumina Hiseq system for 150 nucleotide pair-end sequencing using HiSeq X reagent kit (Catalog: FC-501-2501) and the data was extracted to FastQ format using the Casava software from Illumina