HS treated RNA-seq analysis in Dianthus caryophyllus
Total RNA was extracted from leaves using the Trizol reagent, treated with RNAase-free DNAase and re-purified with the RNeasy kit. Poly(A) mRNA from the total RNA was purified using oligo(dT) beads. Following purification, mRNA was fragmented into small pieces using divalent cations. The first cDNA strand was synthesized using random hexamer primers for reverse transcription with cleaved mRNA fragments serving as templates. The second strand cDNA was synthesized using DNA polymerase I, incubated with RNase H, dNTPs and buffer. Following end repair, ‘A’ bases and adaptors were added to the purified double stranded cDNA fragments. The products are purified and enriched with PCR to create the final cDNA library.