Table 6.

Checklist experimental details to be included in publications.

Step	Parameters to be described
EV isolation
Cell culture	Cell type Confluency Cellular activation/differentiation status Use of EV-depleted serum (include depletion protocol) or serum-free medium Cell viability Mycoplasma test
Body fluid	Health/disease status Collection method (disposables, chemicals, procedure) Removal of body fluid specific contaminants Storage
EV isolation	Differential centrifugation steps Filtration steps Size exclusion chromatography Density gradient Commercially available kits (e.g. chemical, column based) EV quantification method
EV-RNA characterisation
EV-RNA sample preparation	RNase/DNase/proteinase treatment of EV Use of RNA spike RNA isolation method /kit RNA quantification method Bioanalyzer profile of EV-RNA
Library preparation	Enzymatic treatment to remove phosphates, caps, etc. Method /kit for ligating adapters Pre-amplification steps Size selection steps Bioanalyzer profile of library
Sequencing	Platform Maximum read length Direction (paired or single end) Number of cycles Number of replicates (biological or technical)
Bioinformatics	Pre-processing software (trimming/clipping, cut-off values) Reference genome assemblies (release numbers) Primary analysis software (alignment and mapping criteria) Browsers and annotation tools Normalisation methods and software Statistical methods and tools for differential expression analysis
Validation	Validation technique Normalisation procedure; validation of reference genes
Deposition in database	Name of database
EV-RNA functional analysis
EV-RNA transfer	Target cell type and activation/differentiation status Methodology (EV-RNA labelling, target cell RT-qPCR) Quantity of EV per target cell
EV-RNA function	Methodology (transcriptome analysis, functional read-out) Endogenous expression levels of EV-RNA in target cell Proof to attribute functional effects to the RNA component of EV