title: Waus_Transcriptome1
design: To obtain RNA-seq, total RNA was extracted from different samples in TRIzol reagent (Invitrogen, Cat# 15596018)/CTAB-LiCl method (Plant) on dry ice and processed following the manufacturers protocol. Sequencing libraries were generated using TruSeq RNA Library Preparation Kit (Illumina, USA) following standard protocol. Briefly, about 1 g RNA per sample was used and enriched from total RNA using oligo(dT)-attached magnetic beads. The first-strand cDNA was synthesized with random primer and M-MLV Reverse Transcriptase, and the second-strand cDNA synthesis was followed by using DNA Polymerase I and RNase H. The synthesized cDNA was end-repaired, A-tailed and ligated to the sequencing adapters according to library construction protocol. The cDNA fragments were selected by AMPure XP beads (Beckman Coulter, USA) to an average size of 150-200 bp and amplified by PCR with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index Primer. At last, PCR products were purified with AMPure XP Beads (Beckman Coulter, USA) and library quality was assessed on the Agilent Bioanalyzer 2100 system. After that, the library was sequenced on the Illumina NovaSeq 6000 platform.