title: Transcriptome sequencing of MJ treated callus culture-02
design: Illumina-compatible PolyA enriched RNA sequencing library preparation for Frozen Callus RNA was done at Genotypic Technology Pvt. Ltd. Genomics facility. Transcriptome library for sequencing was constructed according to NEBNext Ultra Directional Library Prep Kit (NEB #E7420G, 30061409). 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 adaptor ligation. The adaptor ligated cDNA was cleaned up using HighPrep PCR beads and was subjected to 11 cycles of Indexing-PCR (37  C for 15mins followed by denaturation at 98 C for 30 sec, cycling (98 C for 10sec, 65 C for 75sec) and 65 C for 5mins) to enrich the adaptor-ligated fragments. The PCR product was purified using HighPrep beads. Quantification and size distribution of the prepared libraries were determined using Qubit flourometer (Table 1) and the Agilent D1000 TapeStation respectively according to the manufacturers instructions (Figure 2). The adaptor ligated molecules were quantified on qPCR using KAPA library quantification kit (KAPA Biosystems).