Instrument: Illumina HiSeq 1500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Preparation of Illumina libraries The total RNA libraries were prepared in accordance with the Illumina TruSeq RNA sample preparation v2 guide (Part # 15026495, rev.D, September 2012) for Illumina Paired-End Indexed Sequencing. Step 1: RNA purification and fragmentation According to the Illumina mRNA libraries preparation protocol, poly-A mRNA in the tRNA samples were first purified using Illumina poly-T oligo-attached magnetic beads and two rounds of purification. During the second elution of the poly-A-RNA, the mRNA was also fragmented and primed with random hexamers for cDNA synthesis. Step2: Generation of double-stranded cDNA Cleaved mRNAs were reverse transcribed into first strand cDNA using reverse transcriptase and random primers. The RNA template was then removed and a replacement strand synthesized to generate double-stranded cDNA. Step 3: Preparation of ds cDNA samples for Indexed paired-end sequencing Following the standard protocol, after the first and second strand cDNA synthesis, ends were repaired, dA base added, and Illumina indexing adapters were ligated. Finally, cDNA fragments that have adapter molecules on both ends underwent 15 cylcles of PCR to amplify the amount of prepared material.