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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230672	1/4	Vibrio cholerae	A1552	(d)(U)C DG Filtration	Sjöström AE	2015	56%
Study summary Full title Membrane vesicle-mediated release of bacterial RNA. All authors Sjöström AE, Sandblad L, Uhlin BE, Wai SN Journal Sci Rep Abstract Many Gram-negative bacterial species release outer membrane vesicles (OMVs) that interact with the h (show more...)Many Gram-negative bacterial species release outer membrane vesicles (OMVs) that interact with the host by delivering virulence factors. Here, we report for the first time that RNA is among the wide variety of bacterial components that are associated with OMVs. To characterize the RNA profiles of bacterial OMVs, we performed RNA deep sequencing analysis using OMV samples isolated from a wild type Vibrio cholerae O1 El Tor strain. The results showed that RNAs originating from intergenic regions were the most abundant. Our findings reveal a hitherto unrecognised feature of OMVs mimicking eukaryotic exosomes and highlight a need to evaluate the potential role of RNA-containing bacterial membrane vesicles in bacteria-host interactions. (hide) EV-METRIC 56% (90th 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. For the quantitative method, the reporting of measured EV concentration is expected. 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 Outer membrane vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Filtration Adj. k-factor 0 (pelleting) Protein markers EV: OmpU non-EV: RpoS Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Vibrio cholerae Sample Type Cell culture supernatant EV-producing cells A1552 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 0 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 7 Lowest density fraction 10% Highest density fraction 45% Orientation Top-down Speed (g) 180000 Duration (min) 120 Fraction volume (mL) 0.1-0.3 Fraction processing Centrifugation Pelleting: volume per fraction 0.1-0.3 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 1,70E Pelleting-wash: volume per pellet (mL) 60 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins OmpU Not detected contaminants RpoS Characterization: RNA analysis RNA analysis Type RNA sequencing/ Northern blot Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM/ cryo-EM Image type Close-up, Wide-field Report size (nm) 25-200

	EV230672	2/4	Vibrio cholerae	A1552	(d)(U)C DG Filtration	Sjöström AE	2015	43%
Study summary Full title Membrane vesicle-mediated release of bacterial RNA. All authors Sjöström AE, Sandblad L, Uhlin BE, Wai SN Journal Sci Rep Abstract Many Gram-negative bacterial species release outer membrane vesicles (OMVs) that interact with the h (show more...)Many Gram-negative bacterial species release outer membrane vesicles (OMVs) that interact with the host by delivering virulence factors. Here, we report for the first time that RNA is among the wide variety of bacterial components that are associated with OMVs. To characterize the RNA profiles of bacterial OMVs, we performed RNA deep sequencing analysis using OMV samples isolated from a wild type Vibrio cholerae O1 El Tor strain. The results showed that RNAs originating from intergenic regions were the most abundant. Our findings reveal a hitherto unrecognised feature of OMVs mimicking eukaryotic exosomes and highlight a need to evaluate the potential role of RNA-containing bacterial membrane vesicles in bacteria-host interactions. (hide) EV-METRIC 43% (81st 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. For the quantitative method, the reporting of measured EV concentration is expected. 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 AES206 mutant Focus vesicles Outer membrane vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Filtration Adj. k-factor 0 (pelleting) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Vibrio cholerae Sample Type Cell culture supernatant EV-producing cells A1552 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 0 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 7 Lowest density fraction 10% Highest density fraction 45% Orientation Top-down Speed (g) 180000 Duration (min) 120 Fraction volume (mL) 0.1-0.3 Fraction processing Centrifugation Pelleting: volume per fraction 0.1-0.3 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 1,70E Pelleting-wash: volume per pellet (mL) 60 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: RNA analysis RNA analysis Type RNA sequencing/ Northern blot Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV230672	3/4	Vibrio cholerae	A1552	(d)(U)C DG Filtration	Sjöström AE	2015	43%
Study summary Full title Membrane vesicle-mediated release of bacterial RNA. All authors Sjöström AE, Sandblad L, Uhlin BE, Wai SN Journal Sci Rep Abstract Many Gram-negative bacterial species release outer membrane vesicles (OMVs) that interact with the h (show more...)Many Gram-negative bacterial species release outer membrane vesicles (OMVs) that interact with the host by delivering virulence factors. Here, we report for the first time that RNA is among the wide variety of bacterial components that are associated with OMVs. To characterize the RNA profiles of bacterial OMVs, we performed RNA deep sequencing analysis using OMV samples isolated from a wild type Vibrio cholerae O1 El Tor strain. The results showed that RNAs originating from intergenic regions were the most abundant. Our findings reveal a hitherto unrecognised feature of OMVs mimicking eukaryotic exosomes and highlight a need to evaluate the potential role of RNA-containing bacterial membrane vesicles in bacteria-host interactions. (hide) EV-METRIC 43% (81st 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. For the quantitative method, the reporting of measured EV concentration is expected. 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 AES207 mutant Focus vesicles Outer membrane vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Filtration Adj. k-factor 0 (pelleting) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Vibrio cholerae Sample Type Cell culture supernatant EV-producing cells A1552 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 0 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 7 Lowest density fraction 10% Highest density fraction 45% Orientation Top-down Speed (g) 180000 Duration (min) 120 Fraction volume (mL) 0.1-0.3 Fraction processing Centrifugation Pelleting: volume per fraction 0.1-0.3 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 1,70E Pelleting-wash: volume per pellet (mL) 60 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: RNA analysis RNA analysis Type RNA sequencing/ Northern blot Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV230672	4/4	Vibrio cholerae	A1552	(d)(U)C DG Filtration	Sjöström AE	2015	43%
Study summary Full title Membrane vesicle-mediated release of bacterial RNA. All authors Sjöström AE, Sandblad L, Uhlin BE, Wai SN Journal Sci Rep Abstract Many Gram-negative bacterial species release outer membrane vesicles (OMVs) that interact with the h (show more...)Many Gram-negative bacterial species release outer membrane vesicles (OMVs) that interact with the host by delivering virulence factors. Here, we report for the first time that RNA is among the wide variety of bacterial components that are associated with OMVs. To characterize the RNA profiles of bacterial OMVs, we performed RNA deep sequencing analysis using OMV samples isolated from a wild type Vibrio cholerae O1 El Tor strain. The results showed that RNAs originating from intergenic regions were the most abundant. Our findings reveal a hitherto unrecognised feature of OMVs mimicking eukaryotic exosomes and highlight a need to evaluate the potential role of RNA-containing bacterial membrane vesicles in bacteria-host interactions. (hide) EV-METRIC 43% (81st 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. For the quantitative method, the reporting of measured EV concentration is expected. 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 AES208 mutant Focus vesicles Outer membrane vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Filtration Adj. k-factor 0 (pelleting) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Vibrio cholerae Sample Type Cell culture supernatant EV-producing cells A1552 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 0 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 7 Lowest density fraction 10% Highest density fraction 45% Orientation Top-down Speed (g) 180000 Duration (min) 120 Fraction volume (mL) 0.1-0.3 Fraction processing Centrifugation Pelleting: volume per fraction 0.1-0.3 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 1,70E Pelleting-wash: volume per pellet (mL) 60 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: RNA analysis RNA analysis Type RNA sequencing/ Northern blot Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis None

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EV-TRACK ID	EV230672
species	Vibrio cholerae
sample type	Cell culture
cell type	A1552
condition	Control condition	AES206 mutant	AES207 mutant	AES208 mutant
separation protocol	dUC/ Density gradient/ Filtration	dUC/ Density gradient/ Filtration	dUC/ Density gradient/ Filtration	dUC/ Density gradient/ Filtration
Exp. nr.	1	2	3	4
EV-METRIC %	56	43	43	43