NO2
Library was constructed using TruSeq Stranded RNA LT Sample Prep Kit (Illumina), each prepared from 300 ng of total RNA. The cDNA libraries were prepared according to manufacturer’s instruction as previously described (PMID: 25907828). Briefly, Index codes were ligated to identify individual samples. mRNA was purified from the total RNA using poly-T oligo-attached magnetic beads (Illumina, San Diego, USA), and was then fragmented using divalent cations in the Illumina fragmentation buffer under 94 'C for 1 min. First and second strand cDNAs were synthesized using random oligonucleotides and SuperScript II, followed by DNA polymerase I and RNase H. Overhangs were blunted by using exonuclease/polymerase and, after 3’ end adenylation, Illumina PE adapter oligonucleotides were ligated. DNA fragments that ligated with adaptor molecules on both ends were enriched using the Illumina PCR Primer Cocktail in a 15-cycle PCR reaction. Products were purified and quantified using the AMPure XP and the Agilent Bioanalyzer 2100 systems, respectively. Then the concentration of libraries was quantified using KAPA Library Quantification Kits.