RT3
500 ng of RNA was taken for mRNA isolation, fragmentation and priming. It was further subjected to first strand synthesis followed by second strand synthesis. The dscDNA was purified using HighPrep Beads. Purified cDNA was end-repaired, adenylated and ligated to Illumina multiplex barcode adapters followed by second strand excision using USER enzyme at 37 C for 15mins. Adapter ligated cDNA was purified using HighPrep Beads and was subjected to 11 cycles for Indexing- (98C for 30 sec, cycling (98C for 10sec, 65C for 75sec) and 65C for 5min) to enrich the adapter-ligated fragments. Final PCR product was purified with HighPrep Beads, followed by library quality control. Illumina-compatible sequencing library was quantified by Qubit fluorometer and its fragment size distribution was analyzed.