Instrument: Illumina HiSeq 4000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Root tissue was pulverized in liquid nitrogen using a mortar and pestle, and total RNA was extracted using a RNeasy Plant Mini Kit (Qiagen, Canada) following the manufacturer's protocol. Four biological replicates of Yerong at 0 and 72 h, and of Deder2 at 0, 72, and 120 h time-points, were subjected to RNA-seq analysis. The RNA quality and concentration were verified using a NanoDrop 1000 spectrophotometer (ThermoFisher Scientific, Canada) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Canada) prior to sequencing. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's 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 NEBNext First Strand Synthesis Reaction Buffer （5X）. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase （RNase 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, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. 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 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C 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.