EV uptake experiments

Five thousand human primary fibroblasts (NIGMS Human Genetic Cell Repository #GM08402) were seeded into flat bottom 96-well plates and incubated with 90ul full medium with 10ul PBS-HAT buffer containing 1e5, 1e6, 1e7, 1e8 or 1e9 EVs for 24 hours. Cells were then washed twice with PBS and analyzed. 

RNA-sequencing

Cell or EV RNA was extracted (32) and precipitated as previously described (3) by incubating 500ul TRI Reagent (Sigma), adding 100ul chloroform, and shaking vigorously. After a 15 minute incubation, samples were centrifuged at 12,000 x g for 15 minutes at 4C and 300ul of aqueous phase was mixed with 300ul isopropanol, 30ul of 3M sodium acetate, and 1ul Pellet Paint (Merck) and incubated overnight at –20C. The next morning, samples were centrifuged at 20,000 x g for 30 minutes at 4C, the pellets were washed two times with 700ul of 70% ethanol, before drying and resuspending in 15uL elution buffer (Qiagen). RNA concentrations were measured using Qubit RNA High Sensitivity Assay (Thermo Fischer Scientific) and 2ng were used to generate full length cDNA by Smart-seq2, which utilizes an oligo dT primer (23). 50bp single end reads were sequenced on a HiSeq3000 (Illumina), converted to fastq using bcl2fastq, adapters trimmed using Trim Galore, and the resulting reads aligned to the ENSEMBL human transcriptome GRCh37 or ENSEMBL mouse transcriptome GRCm39 using Tophat 2.1.1. To generate the normalized count matrix, DEseqDataSetFromMatrix and estimateSizeFactors were applied from the DEseq2 package in R (30). 

Clustering, differential expression and gene ontology, and overlap enrichment analysis 

Variable genes above six normalized counts and transcriptome mapping was performed as previously described (28,29). Briefly, Euclidian distances between samples were derived from tSNE analysis using the Rtsne package and maps were assembled by applying force directed connections between each sample and its 25 nearest neighbors for Figure 1-2 and 40 nearest neighbours for Figure 4. Infomap clustering and visualizations were produced using the igraph package in R. Differential expression between groups indicated was performed using the Deseq2 package in R. Genes with an adjusted p-value below 0.05 were separated based on whether they were upregulated or downregulated and analyzed using panther.org complete biological processes statistical overrepresentation test version 10.5281/zenodo.4081749, released 2020-10-09. A control gene set was constructed from all up- and downregulated genes graphed together and the fold enrichments displayed for each individual group were calculated by dividing their enrichment by that of the same term in the control group. Gene overlap enrichment was performed as: (# overlapping genes)/(# genes in group one)*(# genes in group two)(29)