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 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 miRNeasy Mini kit (Qiagen). Briefly previously isolated exosomes were lysed by the addition of Qiazol (Trizol). Then, ethanol was added to the aqueous phase to precipitate the RNA, according to the concentrations indicated by the manufacturer. Samples were also processed by silica columns of RNA isolation specific to the kit, in order to purify the RNA. Finally, 30 μl of RNase free water was added, tempered at 56°C to elute the RNA. From the previously isolated PBMCs, total RNA was also extracted with the same kit, which served as a positive control in PCRs. 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 PEG10 and RNA isolated with Qiagen