GSM3477337: Nef transfection tp2 [miRNA]; Homo sapiens; miRNA-Seq
Exosomes were isolated using PEG precipitation protocol according to Rider et al., 2016 (Rider, M. A. et al. ExtraPEG: A Polyethylene Glycol-Based Method for the Enrichment of Extracellular Vesicles. Sci. Rep. 6, 23978; doi: 10.1038/srep23978 (2016)). Collected conditioned cell culture media was clarified by centrifugation at 500x g, 4oC, 11min to remove lose cells and cellular debris) and by second round of centrifugation at 2000x g, 4oC, 30min to remove cellular debris and large apoptotic bodies. Clarified culture supernatants were mixed with an equal volume of 16% PEG6000 in 1M NaCl and rotated at +4oC overnight. Next day, formed precipitates were sedimented at 3665x g, 4oC for 1hr. The precipitate was dissolved in 12ml cold PBS and the exosomes were pelleted at 100,000 x g for 75 min at 4°C. The exosome pellet was washed with 10 ml of 1X PBS and pelleted again by centrifugation at 100,000 x g for 1 h at 4°C. The resulting pellet was either suspended in 1X PBS for whole exosome applications or further processed for RNA or protein extraction. Total RNA was extracted from exosome pellet using Total Exosome RNA & Protein Isolation Kit (Thermo Fisher, Cat#  4478545) following the manufacturer's protocol. RNA quality and concentration was determined using the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Hewlett-Packard Str. 8, 76337 Waldbronn, Germany). Small RNA libraries were constructed using Illumina TruSeq Small RNA Library Preparation Kit and TruSeq Stranded mRNA kit for Illumina sequencers according to manufacturer's protocol. Individual libraries were constructed using 1µg of total RNA isolated from each sample. Each library was indexed in order to multiplex four samples per sequencing run on the Illumina MiSeq platform using MiSeq 50 cycle Reagent Kits v2.
GEO Accession
GSM3477337