Instrument: Illumina HiSeq 3000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Fresh tissues were homogenized in Rn-easy solution using Bead Mill Homogenizer (Omni International, Kennesaw, GA). Approximately 500ng Total RNA was used for RNA-seq library preparation by following TruSeq stranded mRNA-seq library preparation kit ( Cat. No: 20020595, Illumina, USA). Following mRNA enrichment by oligodT beads, the RNA was fragmented into small pieces using divalent cautions under elevated temperature and magnesium. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. This was followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. Strand specificity was achieved by replacing dTTP with dUTP in the Second Strand Marking Mix (SMM). The incorporation of dUTP in second strand synthesis effectively quenches the second strand during amplification, since the polymerase used in the assay will not incorporate past this nucleotide. These cDNA fragments then went through an end repair process, the addition of a single 'A' base, and then ligation of the adapters. The products were then purified and enriched with PCR to create the final RNA-Seq library for sequencing by 50bp singlle-end protocol with Illumina HiSeq 3000 platform.