RNA-Seq of oryza sativa: acidic stress rice leaf
Total RNA was used to enrich the mRNA using NEBNext Poly (A) mRNA magnetic isolation. The fragmented mRNAs were reverse transcribed to form cDNA. The cDNA reactions were converted to ds DNA. 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 AMPure beads and the DNA is then enriched by PCR with 12 cycles using NEBNext Ultra II Q5 master mix, Illumina universal primer and sample-specific octamer primers. The amplified products were cleaned up by using AM pure beads and the final mRNA library was eluted in 15 uLs of 0.1X TE buffer.