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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV231010	1/4	Equus caballus	Follicular Fluid	(d)(U)C Filtration	Feugang JM	2024	67%
Study summary Full title Seasonal influence on miRNA expression dynamics of extracellular vesicles in equine follicular fluid. All authors Feugang JM, Gad A, Menjivar NG, Ishak GM, Gebremedhn S, Gastal MO, Dlamini NH, Prochazka R, Gastal EL, Tesfaye D Journal J Anim Sci Biotechnol Abstract Ovarian follicular fluid (FF) is a dynamic environment that changes with the seasons, affecting foll (show more...)Ovarian follicular fluid (FF) is a dynamic environment that changes with the seasons, affecting follicle development, ovulation, and oocyte quality. Cells in the follicles release tiny particles called extracellular vesicles (EVs) containing vital regulatory molecules, such as microRNAs (miRNAs). These miRNAs are pivotal in facilitating communication within the follicles through diverse signaling and information transfer forms. EV-coupled miRNA signaling is implicated to be associated with ovarian function, follicle and oocyte growth and response to various environmental insults. Herein, we investigated how seasonal variations directly influence the ovulatory and anovulatory states of ovarian follicles and how are they associated with follicular fluid EV-coupled miRNA dynamics in horses. (hide) EV-METRIC 67% (84th 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 Follicular Fluid Sample origin Anovulatory season Focus vesicles extracellular vesicle 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 Filtration Protein markers EV: CD81/ Flotillin-1/ TSG101 non-EV: Cytochrome C Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Equus caballus Sample Type Follicular Fluid Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 3 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 120000 Filtration steps 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD81/ Flotillin-1/ TSG101 Not detected contaminants Cytochrome C Characterization: RNA analysis RNA analysis Type RNA -sequencing Database NCBI Gene Expression Omnibus (GEO) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 85.9 EV concentration Yes Particle yield particles per milliliter of starting sample: 7.87E+11 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV231010	2/4	Equus caballus	Follicular Fluid	(d)(U)C Filtration	Feugang JM	2024	67%
Study summary Full title Seasonal influence on miRNA expression dynamics of extracellular vesicles in equine follicular fluid. All authors Feugang JM, Gad A, Menjivar NG, Ishak GM, Gebremedhn S, Gastal MO, Dlamini NH, Prochazka R, Gastal EL, Tesfaye D Journal J Anim Sci Biotechnol Abstract Ovarian follicular fluid (FF) is a dynamic environment that changes with the seasons, affecting foll (show more...)Ovarian follicular fluid (FF) is a dynamic environment that changes with the seasons, affecting follicle development, ovulation, and oocyte quality. Cells in the follicles release tiny particles called extracellular vesicles (EVs) containing vital regulatory molecules, such as microRNAs (miRNAs). These miRNAs are pivotal in facilitating communication within the follicles through diverse signaling and information transfer forms. EV-coupled miRNA signaling is implicated to be associated with ovarian function, follicle and oocyte growth and response to various environmental insults. Herein, we investigated how seasonal variations directly influence the ovulatory and anovulatory states of ovarian follicles and how are they associated with follicular fluid EV-coupled miRNA dynamics in horses. (hide) EV-METRIC 67% (84th 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 Follicular Fluid Sample origin Spring ovulatory season Focus vesicles extracellular vesicle 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 Filtration Protein markers EV: CD81/ Flotillin-1/ TSG101 non-EV: Cytochrome C Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Equus caballus Sample Type Follicular Fluid Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 3 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 120000 Filtration steps 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD81/ Flotillin-1/ TSG101 Not detected contaminants Cytochrome C Characterization: RNA analysis RNA analysis Type RNA -sequencing Database NCBI Gene Expression Omnibus (GEO) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 81.2 EV concentration Yes Particle yield particles per milliliter of starting sample: 2.29E+11 EM EM-type Transmission-EM Image type Close-up

	EV231010	3/4	Equus caballus	Follicular Fluid	(d)(U)C Filtration	Feugang JM	2024	67%
Study summary Full title Seasonal influence on miRNA expression dynamics of extracellular vesicles in equine follicular fluid. All authors Feugang JM, Gad A, Menjivar NG, Ishak GM, Gebremedhn S, Gastal MO, Dlamini NH, Prochazka R, Gastal EL, Tesfaye D Journal J Anim Sci Biotechnol Abstract Ovarian follicular fluid (FF) is a dynamic environment that changes with the seasons, affecting foll (show more...)Ovarian follicular fluid (FF) is a dynamic environment that changes with the seasons, affecting follicle development, ovulation, and oocyte quality. Cells in the follicles release tiny particles called extracellular vesicles (EVs) containing vital regulatory molecules, such as microRNAs (miRNAs). These miRNAs are pivotal in facilitating communication within the follicles through diverse signaling and information transfer forms. EV-coupled miRNA signaling is implicated to be associated with ovarian function, follicle and oocyte growth and response to various environmental insults. Herein, we investigated how seasonal variations directly influence the ovulatory and anovulatory states of ovarian follicles and how are they associated with follicular fluid EV-coupled miRNA dynamics in horses. (hide) EV-METRIC 67% (84th 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 Follicular Fluid Sample origin Summer ovulatory season Focus vesicles extracellular vesicle 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 Filtration Protein markers EV: CD81/ Flotillin-1/ TSG101 non-EV: Cytochrome C Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Equus caballus Sample Type Follicular Fluid Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 3 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 120000 Filtration steps 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD81/ Flotillin-1/ TSG101 Not detected contaminants Cytochrome C Characterization: RNA analysis RNA analysis Type RNA -sequencing Database NCBI Gene Expression Omnibus (GEO) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 84.4 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.02E+12 EM EM-type Transmission-EM Image type Close-up

	EV231010	4/4	Equus caballus	Follicular Fluid	(d)(U)C Filtration	Feugang JM	2024	67%
Study summary Full title Seasonal influence on miRNA expression dynamics of extracellular vesicles in equine follicular fluid. All authors Feugang JM, Gad A, Menjivar NG, Ishak GM, Gebremedhn S, Gastal MO, Dlamini NH, Prochazka R, Gastal EL, Tesfaye D Journal J Anim Sci Biotechnol Abstract Ovarian follicular fluid (FF) is a dynamic environment that changes with the seasons, affecting foll (show more...)Ovarian follicular fluid (FF) is a dynamic environment that changes with the seasons, affecting follicle development, ovulation, and oocyte quality. Cells in the follicles release tiny particles called extracellular vesicles (EVs) containing vital regulatory molecules, such as microRNAs (miRNAs). These miRNAs are pivotal in facilitating communication within the follicles through diverse signaling and information transfer forms. EV-coupled miRNA signaling is implicated to be associated with ovarian function, follicle and oocyte growth and response to various environmental insults. Herein, we investigated how seasonal variations directly influence the ovulatory and anovulatory states of ovarian follicles and how are they associated with follicular fluid EV-coupled miRNA dynamics in horses. (hide) EV-METRIC 67% (84th 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 Follicular Fluid Sample origin Fall ovulatory season Focus vesicles extracellular vesicle 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 Filtration Protein markers EV: CD81/ Flotillin-1/ TSG101 non-EV: Cytochrome C Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Equus caballus Sample Type Follicular Fluid Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 3 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 120000 Filtration steps 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD81/ Flotillin-1/ TSG101 Not detected contaminants Cytochrome C Characterization: RNA analysis RNA analysis Type RNA -sequencing Database NCBI Gene Expression Omnibus (GEO) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 79.6 EV concentration Yes Particle yield particles per milliliter of starting sample: 7.93E+11 EM EM-type Transmission-EM Image type Close-up

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EV-TRACK ID	EV231010
species	Equus caballus
sample type	Follicular Fluid
condition	Anovulatory season	Spring ovulatory season	Summer ovulatory season	Fall ovulatory season
separation protocol	dUC/ Filtration	dUC/ Filtration	dUC/ Filtration	dUC/ Filtration
Exp. nr.	1	2	3	4
EV-METRIC %	67	67	67	67