Sahiwal seminal plasma extracellular vesicles (LF3)
RNA sequencing libraries were prepared with Illumina-compatible NEBNext Ultra II Directional RNA Library Prep Kit. 5l of the total RNA was used for fragmentation and priming. Fragmented and primed RNA was further subjected to first strand synthesis followed by second strand synthesis. The double stranded cDNA was purified using NEBNext purification beads. Purified cDNA was end-repaired, adenylated and ligated to Illumina multiplex barcode adapters as per NEBNext Ultra II Directional RNA Library Prep protocol followed by second strand excision using USER enzyme at 37 C for 15mins. Adapter ligated cDNA was purified using NEBNext purification beads and was subjected to12 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 (sequencing library) was purified with JetSeq Beads, followed by library quality control check.