title: Illumina HiSeq 2500 sequencing; Small RNA sequencing of plasma extracellular vesicles from HIV/HCV coinfected patients
design: Small RNA sequencing of plasma extracellular vesicles from HIV/HCV coinfected patients
library_construction_protocol: Whole peripheral blood from four patients were isolated and processed within the first 4 hours. Density gradient centrifugation with Ficoll plaque was performed in order to separate plasma from peripheral blood mononuclear cells (PBMCs). Plasma fraction was then pre-clarified at 15.000rpm 15min at 4°C, four samples pooled and stored at -80°C until use in different aliquots. Plasma was used to isolate extracellular vesicles with a ultracentrifugation (UC) protocol. Briefly Plasma was centrifugated at higher speed (12000g) at first, in order to remove macrovesicles and apoptotic bodies. Afterwards, two ultracentrifuges at 43,000 rpm were performed on a Beckman SW55Ti rotor, on the Beckman Optima L-90K Ultracentrifugue. Finally, the pellet of purified exosomes was resuspended in 100 μl of PBS. The protocol was modified based on the contributions of Rekker et al. (2) and Rani et al.(3), who used another type of rotor, so time and rpm conversions were made according to Beckman Coulter equivalences Total RNA including miRNAs were isolated from plasma and PBMCs using Norgene: Norgene´s Plasma/Serum Exosome and Free-Circulating RNA Isolation Mini Kit which works for isolating any size of RNA from extracellular vesicles such as miRNAs and free-circulating RNAs, were used following manufacturer´s instructions. Finally, RNA was eluted in a volume of 30 µl of the supplied elution buffer.  Small RNA library was performed with a high sensitivity kit for miRNA analysis in exosomes, the TailorMix miRNA Sample Preparation Kit (SeqMatic, ref. TM302), according to the manufacturer's protocol
attributes.tag: Experimental Factor: protocol
attributes.value: Exosome isolated with UC and RNA isolated with Norgene