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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	separation protocol	First author	Year	EV-METRIC
	EV170030	1/1	Mus musculus	Cell culture supernatant	DG (d)(U)C	Driedonks, Tom A. P.	2018	100%
Study summary Full title Immune stimuli shape the small non-coding transcriptome of extracellular vesicles released by dendritic cells. All authors Driedonks TAP, van der Grein SG, Ariyurek Y, Buermans HPJ, Jekel H, Chow FWN, Wauben MHM, Buck AH, 't Hoen PAC, Nolte-'t Hoen ENM Journal Cell Mol Life Sci Abstract The release and uptake of nano-sized extracellular vesicles (EV) is a highly conserved means of inte (show more...)The release and uptake of nano-sized extracellular vesicles (EV) is a highly conserved means of intercellular communication. The molecular composition of EV, and thereby their signaling function to target cells, is regulated by cellular activation and differentiation stimuli. EV are regarded as snapshots of cells and are, therefore, in the limelight as biomarkers for disease. Although research on EV-associated RNA has predominantly focused on microRNAs, the transcriptome of EV consists of multiple classes of small non-coding RNAs with potential gene-regulatory functions. It is not known whether environmental cues imposed on cells induce specific changes in a broad range of EV-associated RNA classes. Here, we investigated whether immune-activating or -suppressing stimuli imposed on primary dendritic cells affected the release of various small non-coding RNAs via EV. The small RNA transcriptomes of highly pure EV populations free from ribonucleoprotein particles were analyzed by RNA sequencing and RT-qPCR. Immune stimulus-specific changes were found in the miRNA, snoRNA, and Y-RNA content of EV from dendritic cells, whereas tRNA and snRNA levels were much less affected. Only part of the changes in EV-RNA content reflected changes in cellular RNA, which urges caution in interpreting EV as snapshots of cells. By comprehensive analysis of RNA obtained from highly purified EV, we demonstrate that multiple RNA classes contribute to genetic messages conveyed via EV. The identification of multiple RNA classes that display cell stimulation-dependent association with EV is the prelude to unraveling the function and biomarker potential of these EV-RNAs. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + (d)(U)C Adj. k-factor 253.9 (pelleting) Protein markers EV: MHC2/ CD63/ CD9/ Galectin-3 non-EV: beta-actin Proteomics no Show all info Study aim Biomarker, Biogenesis/cargo sorting, Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells primary bone marrow dendritic cells EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Cell viability 90 Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 65 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Density gradient Density medium Sucrose Type Continuous Lowest density fraction 0.4M Highest density fraction 2.5M Sample volume (mL) 0.15 Orientation Bottom-up (sample migrates upwards) Rotor type SW 40 Ti Speed (g) 192000 Duration (min) 900-1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 1 Pelleting: duration (min) 65 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 192000 Pelleting: adjusted k-factor 144.0 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD63, MHC2, Galectin-3 Not detected contaminants beta-actin Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100 - 200 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type BD Influx Hardware adjustment see van der Vlist et al. 2012 Nature Protocols;and Nolte-'t Hoen 2012 Nanomedicine Calibration bead size 0.1,0.2 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

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