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 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 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 TE and RNA isolated with Qiagen