Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: RNAeasy column (Qiagen) mRNA was enriched using oligo(dT) beads; mRNA was then fragmented randomly in fragmentation buffer, followed by cDNA synthesis using random hexamers and reverse transcriptase. After first-strand synthesis, a custom second-strand synthesis buffer (Illumina) was added with dNTPs, RNase H and Escherichia coli polymerase I to generate the second strand by nick-translation. Then a round of purification, terminal repair, A-tailing, ligation of sequencing adapters, size selection and PCR enrichment was performed. Library concentration was first quantified using a Qubit 2.0 fluorometer (Life Technologies), and then diluted to 1 ng/μl before checking insert size on an Agilent 2100 and quantifying to greater accuracy by quantitative PCR (Q-PCR) (library activity >2 nM). Libraries were then fed into HiSeq2000 machines according to activity and expected data volume. quantifying to greater accuracy by quantitative PCR (Q-PCR) (library activity >2 nM).