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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV170067	1/6	Homo sapiens	HMC-1	DG (d)(U)C	Lässer C	2017	78%
Study summary Full title Two distinct extracellular RNA signatures released by a single cell type identified by microarray and next-generation sequencing. All authors Lässer C, Shelke GV, Yeri A, Kim DK, Crescitelli R, Raimondo S, Sjöstrand M, Gho YS, Van Keuren Jensen K, Lötvall J. Journal RNA Biol Abstract Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in (show more...)Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in many body fluids such as blood, breast milk and cerebrospinal fluid. However, there are conflicting results regarding the nature of exRNA. Here, we have separated 2 distinct exRNA profiles released by mast cells, here termed high-density (HD) and low-density (LD) exRNA. The exRNA in both fractions was characterized by microarray and next-generation sequencing. Both exRNA fractions contained mRNA and miRNA, and the mRNAs in the LD exRNA correlated closely with the cellular mRNA, whereas the HD mRNA did not. Furthermore, the HD exRNA was enriched in lincRNA, antisense RNA, vault RNA, snoRNA, and snRNA with little or no evidence of full-length 18S and 28S rRNA. The LD exRNA was enriched in mitochondrial rRNA, mitochondrial tRNA, tRNA, piRNA, Y RNA, and full-length 18S and 28S rRNA. The proteomes of the HD and LD exRNA-containing fractions were determined with LC-MS/MS and analyzed with Gene Ontology term finder, which showed that both proteomes were associated with the term extracellular vesicles and electron microscopy suggests that at least a part of the exRNA is associated with exosome-like extracellular vesicles. Additionally, the proteins in the HD fractions tended to be associated with the nucleus and ribosomes, whereas the LD fraction proteome tended to be associated with the mitochondrion. We show that the 2 exRNA signatures released by a single cell type can be separated by floatation on a density gradient. These results show that cells can release multiple types of exRNA with substantial differences in RNA species content. This is important for any future studies determining the nature and function of exRNA released from different cells under different conditions. (hide) EV-METRIC 78% (97th 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 Extracellular vesicle-like structures 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 DG (d)(U)C Protein markers EV: TSG101/ CD63/ CD81/ Flotillin1/ ANXA2/ CD9 non-EV: Proteomics yes EV density (g/ml) 1.09 - 1.31 Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HMC-1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 120000 Density gradient Type Discontinuous Number of initial discontinuous layers 16 Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 35 Sample volume (mL) 2 Orientation Bottom-up Rotor type SW 32 Ti Speed (g) 175000 Duration (min) 960 Fraction volume (mL) 3.9 Fraction processing Centrifugation Pelleting: duration (min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 120000 EV-subtype Distinction between multiple subtypes Density Used subtypes 1.09-1.21 g/mL Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1/ ANXA2/ TSG101/ CD81 Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins CD9/ CD63/ CD81 Proteomics database Yes Characterization: RNA analysis RNA analysis Type RNA sequencing;Microarray Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes EM EM-type Immuno-EM EM protein CD63 Image type Close-up, Wide-field Report size (nm) 92

	EV170067	2/6	Homo sapiens	HMC-1	DG (d)(U)C Filtration	Lässer C	2017	67%
Study summary Full title Two distinct extracellular RNA signatures released by a single cell type identified by microarray and next-generation sequencing. All authors Lässer C, Shelke GV, Yeri A, Kim DK, Crescitelli R, Raimondo S, Sjöstrand M, Gho YS, Van Keuren Jensen K, Lötvall J. Journal RNA Biol Abstract Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in (show more...)Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in many body fluids such as blood, breast milk and cerebrospinal fluid. However, there are conflicting results regarding the nature of exRNA. Here, we have separated 2 distinct exRNA profiles released by mast cells, here termed high-density (HD) and low-density (LD) exRNA. The exRNA in both fractions was characterized by microarray and next-generation sequencing. Both exRNA fractions contained mRNA and miRNA, and the mRNAs in the LD exRNA correlated closely with the cellular mRNA, whereas the HD mRNA did not. Furthermore, the HD exRNA was enriched in lincRNA, antisense RNA, vault RNA, snoRNA, and snRNA with little or no evidence of full-length 18S and 28S rRNA. The LD exRNA was enriched in mitochondrial rRNA, mitochondrial tRNA, tRNA, piRNA, Y RNA, and full-length 18S and 28S rRNA. The proteomes of the HD and LD exRNA-containing fractions were determined with LC-MS/MS and analyzed with Gene Ontology term finder, which showed that both proteomes were associated with the term extracellular vesicles and electron microscopy suggests that at least a part of the exRNA is associated with exosome-like extracellular vesicles. Additionally, the proteins in the HD fractions tended to be associated with the nucleus and ribosomes, whereas the LD fraction proteome tended to be associated with the mitochondrion. We show that the 2 exRNA signatures released by a single cell type can be separated by floatation on a density gradient. These results show that cells can release multiple types of exRNA with substantial differences in RNA species content. This is important for any future studies determining the nature and function of exRNA released from different cells under different conditions. (hide) EV-METRIC 67% (94th 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 Extracellular vesicle-like structures 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 DG (d)(U)C Filtration Protein markers EV: TSG101/ CD63/ CD81/ Flotillin1/ ANXA2/ CD9 non-EV: Proteomics yes EV density (g/ml) 1.09-1.31 Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HMC-1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 120000 Density gradient Type Discontinuous Number of initial discontinuous layers 16 Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 35 Sample volume (mL) 2 Orientation Bottom-up Rotor type SW 32 Ti Speed (g) 175000 Duration (min) 960 Fraction volume (mL) 3.9 Fraction processing Centrifugation Pelleting: duration (min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 120000 Filtration steps 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes Density Used subtypes 1.24-1.31 g/mL Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1/ TSG101/ CD81 Not detected EV-associated proteins ANXA2 Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins CD9/ CD63/ CD81 Proteomics database Yes Characterization: RNA analysis RNA analysis Type RNA sequencing;Microarray Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes EM EM-type Immuno-EM EM protein CD63 Image type Wide-field Report size (nm) 51

	EV170067	3/6	Homo sapiens	HMC-1	DG (d)(U)C	Lässer C	2017	57%
Study summary Full title Two distinct extracellular RNA signatures released by a single cell type identified by microarray and next-generation sequencing. All authors Lässer C, Shelke GV, Yeri A, Kim DK, Crescitelli R, Raimondo S, Sjöstrand M, Gho YS, Van Keuren Jensen K, Lötvall J. Journal RNA Biol Abstract Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in (show more...)Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in many body fluids such as blood, breast milk and cerebrospinal fluid. However, there are conflicting results regarding the nature of exRNA. Here, we have separated 2 distinct exRNA profiles released by mast cells, here termed high-density (HD) and low-density (LD) exRNA. The exRNA in both fractions was characterized by microarray and next-generation sequencing. Both exRNA fractions contained mRNA and miRNA, and the mRNAs in the LD exRNA correlated closely with the cellular mRNA, whereas the HD mRNA did not. Furthermore, the HD exRNA was enriched in lincRNA, antisense RNA, vault RNA, snoRNA, and snRNA with little or no evidence of full-length 18S and 28S rRNA. The LD exRNA was enriched in mitochondrial rRNA, mitochondrial tRNA, tRNA, piRNA, Y RNA, and full-length 18S and 28S rRNA. The proteomes of the HD and LD exRNA-containing fractions were determined with LC-MS/MS and analyzed with Gene Ontology term finder, which showed that both proteomes were associated with the term extracellular vesicles and electron microscopy suggests that at least a part of the exRNA is associated with exosome-like extracellular vesicles. Additionally, the proteins in the HD fractions tended to be associated with the nucleus and ribosomes, whereas the LD fraction proteome tended to be associated with the mitochondrion. We show that the 2 exRNA signatures released by a single cell type can be separated by floatation on a density gradient. These results show that cells can release multiple types of exRNA with substantial differences in RNA species content. This is important for any future studies determining the nature and function of exRNA released from different cells under different conditions. (hide) EV-METRIC 57% (92nd 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 Extracellular vesicle-like structures 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 DG (d)(U)C Protein markers EV: non-EV: Proteomics no EV density (g/ml) 1.09 - 1.31 Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HMC-1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 120000 Density gradient Type Discontinuous Number of initial discontinuous layers 16 Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 35 Sample volume (mL) 2 Orientation Top-down Rotor type SW 32 Ti Speed (g) 175000 Duration (min) 960 Fraction volume (mL) 3.9 Fraction processing Centrifugation Pelleting: duration (min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 120000 EV-subtype Distinction between multiple subtypes Density Used subtypes 1.09 - 1.21 g/mL Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: RNA analysis RNA analysis Type RNA sequencing,Microarray Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No EM EM-type Immuno-EM EM protein CD63 Image type Close-up, Wide-field Report size (nm) 92

	EV170067	4/6	Homo sapiens	HMC-1	DG (d)(U)C Filtration	Lässer C	2017	43%
Study summary Full title Two distinct extracellular RNA signatures released by a single cell type identified by microarray and next-generation sequencing. All authors Lässer C, Shelke GV, Yeri A, Kim DK, Crescitelli R, Raimondo S, Sjöstrand M, Gho YS, Van Keuren Jensen K, Lötvall J. Journal RNA Biol Abstract Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in (show more...)Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in many body fluids such as blood, breast milk and cerebrospinal fluid. However, there are conflicting results regarding the nature of exRNA. Here, we have separated 2 distinct exRNA profiles released by mast cells, here termed high-density (HD) and low-density (LD) exRNA. The exRNA in both fractions was characterized by microarray and next-generation sequencing. Both exRNA fractions contained mRNA and miRNA, and the mRNAs in the LD exRNA correlated closely with the cellular mRNA, whereas the HD mRNA did not. Furthermore, the HD exRNA was enriched in lincRNA, antisense RNA, vault RNA, snoRNA, and snRNA with little or no evidence of full-length 18S and 28S rRNA. The LD exRNA was enriched in mitochondrial rRNA, mitochondrial tRNA, tRNA, piRNA, Y RNA, and full-length 18S and 28S rRNA. The proteomes of the HD and LD exRNA-containing fractions were determined with LC-MS/MS and analyzed with Gene Ontology term finder, which showed that both proteomes were associated with the term extracellular vesicles and electron microscopy suggests that at least a part of the exRNA is associated with exosome-like extracellular vesicles. Additionally, the proteins in the HD fractions tended to be associated with the nucleus and ribosomes, whereas the LD fraction proteome tended to be associated with the mitochondrion. We show that the 2 exRNA signatures released by a single cell type can be separated by floatation on a density gradient. These results show that cells can release multiple types of exRNA with substantial differences in RNA species content. This is important for any future studies determining the nature and function of exRNA released from different cells under different conditions. (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 Control condition Focus vesicles Extracellular vesicle-like structures 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 DG (d)(U)C Filtration Protein markers EV: non-EV: Proteomics no EV density (g/ml) 1.09 - 1.31 Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HMC-1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 120000 Density gradient Type Discontinuous Number of initial discontinuous layers 16 Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 35 Sample volume (mL) 2 Orientation Top-down Rotor type SW 32 Ti Speed (g) 175000 Duration (min) 960 Fraction volume (mL) 3.9 Fraction processing Centrifugation Pelleting: duration (min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 120000 Filtration steps 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes Density Used subtypes 1.24 - 1.31 g/mL Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: RNA analysis RNA analysis Type RNA sequencing;Microarray Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No EM EM-type Immuno-EM EM protein CD63 Image type Wide-field Report size (nm) 51

	EV170067	5/6	Homo sapiens	TF-1	DG (d)(U)C Filtration	Lässer C	2017	43%
Study summary Full title Two distinct extracellular RNA signatures released by a single cell type identified by microarray and next-generation sequencing. All authors Lässer C, Shelke GV, Yeri A, Kim DK, Crescitelli R, Raimondo S, Sjöstrand M, Gho YS, Van Keuren Jensen K, Lötvall J. Journal RNA Biol Abstract Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in (show more...)Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in many body fluids such as blood, breast milk and cerebrospinal fluid. However, there are conflicting results regarding the nature of exRNA. Here, we have separated 2 distinct exRNA profiles released by mast cells, here termed high-density (HD) and low-density (LD) exRNA. The exRNA in both fractions was characterized by microarray and next-generation sequencing. Both exRNA fractions contained mRNA and miRNA, and the mRNAs in the LD exRNA correlated closely with the cellular mRNA, whereas the HD mRNA did not. Furthermore, the HD exRNA was enriched in lincRNA, antisense RNA, vault RNA, snoRNA, and snRNA with little or no evidence of full-length 18S and 28S rRNA. The LD exRNA was enriched in mitochondrial rRNA, mitochondrial tRNA, tRNA, piRNA, Y RNA, and full-length 18S and 28S rRNA. The proteomes of the HD and LD exRNA-containing fractions were determined with LC-MS/MS and analyzed with Gene Ontology term finder, which showed that both proteomes were associated with the term extracellular vesicles and electron microscopy suggests that at least a part of the exRNA is associated with exosome-like extracellular vesicles. Additionally, the proteins in the HD fractions tended to be associated with the nucleus and ribosomes, whereas the LD fraction proteome tended to be associated with the mitochondrion. We show that the 2 exRNA signatures released by a single cell type can be separated by floatation on a density gradient. These results show that cells can release multiple types of exRNA with substantial differences in RNA species content. This is important for any future studies determining the nature and function of exRNA released from different cells under different conditions. (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 Control condition Focus vesicles Extracellular vesicle-like structures 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 DG (d)(U)C Filtration Protein markers EV: non-EV: Proteomics no EV density (g/ml) 1.09 - 1.31 Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells TF-1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 120000 Density gradient Type Discontinuous Number of initial discontinuous layers 16 Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 35 Sample volume (mL) 2 Orientation Bottom-up Rotor type SW 32 Ti Speed (g) 175000 Duration (min) 960 Fraction volume (mL) 3.9 Fraction processing Centrifugation Pelleting: duration (min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 120000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: RNA analysis RNA analysis Type Other Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No

	EV170067	6/6	Homo sapiens	TF-1	DG (d)(U)C Filtration	Lässer C	2017	43%
Study summary Full title Two distinct extracellular RNA signatures released by a single cell type identified by microarray and next-generation sequencing. All authors Lässer C, Shelke GV, Yeri A, Kim DK, Crescitelli R, Raimondo S, Sjöstrand M, Gho YS, Van Keuren Jensen K, Lötvall J. Journal RNA Biol Abstract Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in (show more...)Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in many body fluids such as blood, breast milk and cerebrospinal fluid. However, there are conflicting results regarding the nature of exRNA. Here, we have separated 2 distinct exRNA profiles released by mast cells, here termed high-density (HD) and low-density (LD) exRNA. The exRNA in both fractions was characterized by microarray and next-generation sequencing. Both exRNA fractions contained mRNA and miRNA, and the mRNAs in the LD exRNA correlated closely with the cellular mRNA, whereas the HD mRNA did not. Furthermore, the HD exRNA was enriched in lincRNA, antisense RNA, vault RNA, snoRNA, and snRNA with little or no evidence of full-length 18S and 28S rRNA. The LD exRNA was enriched in mitochondrial rRNA, mitochondrial tRNA, tRNA, piRNA, Y RNA, and full-length 18S and 28S rRNA. The proteomes of the HD and LD exRNA-containing fractions were determined with LC-MS/MS and analyzed with Gene Ontology term finder, which showed that both proteomes were associated with the term extracellular vesicles and electron microscopy suggests that at least a part of the exRNA is associated with exosome-like extracellular vesicles. Additionally, the proteins in the HD fractions tended to be associated with the nucleus and ribosomes, whereas the LD fraction proteome tended to be associated with the mitochondrion. We show that the 2 exRNA signatures released by a single cell type can be separated by floatation on a density gradient. These results show that cells can release multiple types of exRNA with substantial differences in RNA species content. This is important for any future studies determining the nature and function of exRNA released from different cells under different conditions. (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 Control condition Focus vesicles Extracellular vesicle-like structures 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 DG (d)(U)C Filtration Protein markers EV: non-EV: Proteomics no EV density (g/ml) 1.09 - 1.31 Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells TF-1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 120000 Density gradient Type Discontinuous Number of initial discontinuous layers 16 Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 35 Sample volume (mL) 2 Orientation Bottom-up Rotor type SW 32 Ti Speed (g) 175000 Duration (min) 960 Fraction volume (mL) 3.9 Fraction processing Centrifugation Pelleting: duration (min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 120000 Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: RNA analysis RNA analysis Type Other Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No

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EV-TRACK ID	EV170067
species	Homo sapiens
sample type	Cell culture
cell type	HMC-1	HMC-1	HMC-1	HMC-1	TF-1	TF-1
condition	Control condition	Control condition	Control condition	Control condition	Control condition	Control condition
separation protocol	DG (d)(U)C	DG (d)(U)C Filtration	DG (d)(U)C	DG (d)(U)C Filtration	DG (d)(U)C Filtration	DG (d)(U)C Filtration
EV subtype	1.09-1.21 g/mL	1.24-1.31 g/mL	1.09 - 1.21 g/mL	1.24 - 1.31 g/mL	NA	NA
Exp. nr.	1	2	3	4	5	6
EV-METRIC %	78	67	57	43	43	43