title: RNA-seq of Arabidopsis thaliana: silique
design: Approximate 100 mg of plant tissues were grounded and treated by TRIzol, and ~200-2000 ng/ľl of total RNA were generated. RNA sequencing libraries were made using the ŇNEBNext¨ UltraŞ Directional RNA Library Prep Kit for IlluminaÓ according to the manufactureŐs protocol. Specifically, mRNA was isolated using the NEBNext Oligo d(T)26 and was then fragmented at 94_ for 15 min. First and second cDNA were sequentially synthesized in succession. After end repairing and dA-tailing of the cDNA library, the multiplex adaptors (Index Primers Set 1 and Set 2) were ligated to the two ends of the cDNAs. Finally, each library was enriched by 14 cycles of amplification. The quality of cDNA libraries with the multiplex adaptors was checked using Bioanalyzer (Aglient), and the quantity of cDNA was measured using Qubit (Invitrogen). High quality libraries were then pooled based on the balance of the concentration of each library. Fifteen libraries were mixed into one pool, and each library had a different oligo sequence locating the downstream side of the adaptor. The loading volume of the pooled libraries was decided using real-time polymerase chain reaction. All libraries were sequenced in one lane of Illumina HiSeq run and then separated by the corresponding tag sequences.