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 polyethylene glycol (PEG) of 10 KDa. Briefly, 50% PEG 10KDawas used in deionized water (Weng et al., 2016). 250 μl of PEG was added to 1 ml of plasma, incubated 2h at 4°C and centrifuged at 3000g and at 4°C. Finally, the pellet was resuspended in 200 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 PEG10 and RNA isolated with Norgene