title: RNA-Seq of  Hippophae rhamnoides L. subsp. Sinensis Rousi: adult female leaves
design: A total amount of 1.5  g RNA per sample was used as input material for the RNA sample preparations. 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 250~300 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 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.