title: TranscriptomesequencingofE.prostratashoot-02
design: Illumina-compatible PolyA enriched RNA sequencing library preparation for Brassica juncea RNA was performed at Genotypic Technology Pvt. Ltd. Genomics facility.Transcriptome library for sequencing was constructed according to NEBNext Ultra Directional Library Prep Kit (NEB, #E7420S). 500ng of Qubit quantified Total RNA was taken for mRNA isolation, fragmentation and priming. The fragmented and primed mRNA was further subjected to first and second strand synthesis. The double stranded cDNA was cleaned up using HighPrep PCR (Magbio, Cat #AC-60050) beads. Subsequently, cDNA was subjected to End Repair, A Tailing followed by adapter ligation. The adapter ligated cDNA was cleaned up using HighPrep PCR beads and was followed by 12 cycles of Indexing-PCR (37 C for 15mins followed by denaturation at 98C for 30 sec, cycling (98C for 10sec, 65C for 75sec) and 65C for 5mins) to enrich the adapter-ligated fragments. The PCR product was purified using HighPrep beads. Quantification and size distribution of the prepared libraries were determined using Qubit flourometer and the Agilent D1000 TapeStation respectively according to the manufacturers instructions. The adapter ligated molecules were quantified on qPCR using KAPA library quantification kit (KAPA Biosystems).