Illumina HiSeq 2500 sequencing; Small RNA sequencing of plasma extracellular vesicles from HIV/HCV coinfected patients
Small RNA sequencing of plasma extracellular vesicles from HIV/HCV coinfected patients
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 Total exosome isolation kit (TE)(Thermofisher). The kit was used according to the manufacturer´s instructions, with modifications recommended by Lane (2015) and Van Deun (2014). Proteinase K was added to the clarified plasma by incubating it at 37°C, so as to eliminate plasma proteins, maximize the purity of the exosomes and dissociate the extracellular RNAs (exRNAs) of protein complexes. Finally, EPR (exosome precipitation reagent) was added to precipitate the exosomes. It was incubated at 4°C overnight and the pellet was resuspended in 100 μl of PBS. 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
Experimental Factor: protocol
Exosome isolated with TE and RNA isolated with Norgene