title: RNA-Seq of S. indicum (Control)
design: For RNA-seq, total RNA was isolated from leaves using Spectrum Plant Total RNA kit (SIGMA). RNA integrity (RIN) and qunatitation was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technology, USA). A total amount of 1 g RNA per sample was used as input material for the RNA sample preparations. Total RNA was processed using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB E7490) and six libraries were prepared with the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) following manufacturers instruction (E7530). Index codes were added to attribute sequences to each sample. After the quality check procedures, mRNA from total RNA was enriched using oligo(dT) beads. The mRNA was then fragmented randomly in NEBNext First Strand Synthesis Reaction Buffer (5X) using di-valent cations under elevated temperature. First strand of cDNA was synthesized using Random hexamer primer and M-MuLV Reverse Transcriptase (RNase-H). After first-strand synthesis, a custom second-strand synthesis buffer (Illumina) is added, with dNTPs, RNase H and DNA polymerase I to generate the second strand by nick-translation. 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 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 clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina) according to the manufacturers instructions. After cluster generation, six library preparations were sequenced on an Illumina platform (Illumina NextSeq. 1000) and paired-end reads were generated.