alias: GSM2212222
title: GSM2212222: MDA-MB-231control-cell; Homo sapiens; miRNA-Seq
library_construction_protocol: Exosomes were isolated utilizing a combination of centrifugation, ultracentrifugation, and filtration as we have previously described [16] or with the Exoquick-TC reagent (System Biosciences, Mountain View, CA) following the manufacturer’s protocol. For ultracentrifugation isolation, conditioned cell culture media was collected and centrifuged at 10,000 x g for 30 min at 4°C, to remove cells and large debris. The supernatant was filtered using a 0.22-μm pore filter and the exosomes were pelleted at 100,000 x g for 1 h 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 the TRIzol reagent (Invitrogen/Life Technologies) following the manufacturer’s protocol. RNA concentration was quantitated using the NanoDrop ND-100 Spectrophotometer (NanoDrop Technologies, Wilmington, DE). Small RNA libraries were constructed using New England Biolabs (NEB) NEBNext Multiplex Small RNA Library Prep Set for Illumina sequencers and the NEB standard protocol. Individual libraries were constructed using 1µg of total RNA isolated from each sample.
identifiers.namespace: GEO
identifiers.id: GSM2212222