root2
A total amount of 2 g RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using VAHTSTM mRNA-seq V2 Library Prep Kit for Illumina following manufacturers recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in VAHTSTM First Strand Synthesis Reaction Buffer5X. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse TranscriptaseRNase H-. Second strand cDNA synthesis was subsequently performed using DNA polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3 ends of DNA fragments, Adaptor were ligated to prepare for library. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 l USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37C for 15 min followed by 5 min at 95 C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The libraries were then quantified and pooled. Paired-end sequencing of the library was performed on the HiSeq XTen sequencers (Illumina, San Diego, CA).