6h 2
Total RNA were extracted from the stem of B. sacra plant collected after 0, 30min, 3hours and 6hours of wounding stress (two biological replicates), using the Pure Link Plant RNA reagent kit (Life Technologies, USA) with some modifications and in-house developed HMW RNA extraction method. The cDNA libraries were prepared for the ribominus total RNA were constructed using the Ion total RNA-seq kit V2, ERCC RNA spike- In control mixes were added according to the manufacturer protocol for 100ng. The total RNA were fragmented using RNases III followed by the purification, the yield was checked on the Bioanalyzer, followed by the amplification of cDNA, the yield was checked on the Bioanalyzer. The Ion One Touch 2 system (Life Technologies, USA) was used for the emulsion PCR of cDNA, which was followed by the Ion One Touch ES for the enrichment of template. The enriched template was loaded into Ion 540 Chips for the transcriptome sequencing on Ion Torrent S5.