Supplementary Information Heterogeneity and interplay of the extracellular vesicle small RNA transcriptome and proteome Helena Sork, Giulia Corso, Kaarel Krjutskov, Henrik J. Johansson, Joel Z. Nordin, Oscar P.B. Wiklander, Yi Xin Fiona Lee, Jakub Orzechowski Westholm, Janne Lehtiö, Matthew J.A. Wood, Imre Mäger, Samir EL Andaloussi cells EVs cells EVs ALIX (96 kDa) TSG101 (50 kDa) cells EVs cells EVs Calnexin (75 kDa) SDCBP (32 kDa) cells EVs ß-actin (42 kDa) Supplementary Figure S1. Uncropped western blot images of cell- and EV samples. Cropped images can be found in Figure 1a. SDCBP (syntenin, human reactive antibody). 17-35nt RNA sequences ❶ ‘small RNA’ annotation a) miRNA. piRNA. snRNA. and snoRNA b) No feature ❷ ‘rRNA’ annotation a) LSU/SSU rRNA. Mt_rRNA. predicted rRNA (MMu). rRNA pseudogenes (HSa) b) No feature ❸ ‘other RNA’ annotation a) tRNA. Mt_tRNA. protein coding genes. lncRNA. misc_RNA. proc_transcripts. pseudogenes and scRNA b) No feature Supplementary Figure S2. Annotation of small RNA sequencing data. All mapped reads in the size range of 17–35 nt reads were annotated against the respective human or mouse ‘small RNA’ genomic features (gene biotypes according to Vega, version 68 ). The reads with no found ‘small RNA’ feature were filtered out and subjected to ’rRNA’ annotation. Finally, all reads that did not match ‘small RNA’ or ‘rRNA’ were annotated against the custom-compiled list of ‘other RNA’ annotations. LSU/SSU rRNA – large/small subunit ribosomal RNA; Mt_rRNA – mitochondrial ribosomal RNA; misc_RNA – miscalleous RNA (includes Y RNA); proc_transcripts – processed transcripts. scRNA - small cytoplasmic RNA. a C E L L S E V s 6 0 6 0 ) % 5 0 5 0 H E K ( t n 4 0 4 0 R D 4 u o c 3 0 3 0 C 2 C 1 2 d a e N e u r o 2 a r 2 0 2 0 n a C 1 7 .2 e 1 0 1 0 M 0 0 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 3 0 3 1 3 2 3 3 3 4 3 5 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 3 0 3 1 3 2 3 3 3 4 3 5 r e a d le n g t h ( n t ) b H E K c R D 4 d C 2 C 1 2 1 0 6 1 0 6 1 0 6 2 2 2 R = 0 .8 6 1 0 R = 0 .8 1 2 8 R = 0 .9 8 2 5 1 0 5 1 0 5 1 0 5 d d d 4 4 4 a a a 1 0 1 0 1 0 e e e s s s r r r V V V 3 3 3 d d d 1 0 1 0 1 0 E E E e e e , , , t t t s s s 2 2 2 i i i 1 0 1 0 1 0 l l l n n n a a a u u u 1 1 1 o o o m m m 1 0 1 0 1 0 r r r c c c o o o 0 0 0 N N N 1 0 1 0 1 0 n d n d n d n d 1 0 0 1 0 1 1 0 2 1 0 3 1 0 4 1 0 5 1 0 6 n d 1 0 0 1 0 1 1 0 2 1 0 3 1 0 4 1 0 5 1 0 6 n d 1 0 0 1 0 1 1 0 2 1 0 3 1 0 4 1 0 5 1 0 6 N o rm a lis e d re a d N o rm a lis e d re a d N o rm a lis e d re a d c o u n t, c e lls c o u n t, c e lls c o u n t, c e lls HUMAN e N e u r o 2 a f C 1 7 .2 g HEK cells 1 0 6 1 0 6 2 2 R = 0 .8 7 2 6 R = 0 .9 5 1 1 HEK EVs 5 5 1 0 1 0 RD4 cells d d 4 4 a 1 0 a 1 0 e e s s RD4 EVs r r V 3 V 3 d 1 0 d 1 0 E E e e , , t s t 2 s 2 i MOUSE 1 0 i l 1 0 l n n a a u u 1 1 C2C12 cells o o m 1 0 m 1 0 r c r c o o C2C12 EVs 0 0 N 1 0 N 1 0 C17.2 cells n d n d C17.2 EVs n d 1 0 0 1 0 1 1 0 2 1 0 3 1 0 4 1 0 5 1 0 6 n d 1 0 0 1 0 1 1 0 2 1 0 3 1 0 4 1 0 5 1 0 6 Neuro2a cells N o rm a lis e d re a d N o rm a lis e d re a d Neuro2a EVs c o u n t, c e lls c o u n t, c e lls h i row max row min row max row min Supplementary Figure S3. Length distribution, correlation and clustering of vesicular and cellular miRNAs. (a) Length distribution of miRNA reads indicates to the prominence of mature miRNA sequences. Normalised miRNA read count in HEK (b), RD4 (c), C2C12 (d), Neuro2a (e) and C17.2 (f) miRNA expression levels in EVs correlate with levels in parental cells. A small number of individual miRNAs deviated from the correlation (few selected samples are labelled in panels A-E). (g) Hierarchical clustering of log2 transformed normalized miRNA counts showed that EVs cluster together with their parental cells rather than with EVs derived from other cell types. (h-i) Heatmaps of top 10 EV-enriched miRNAs within human (h) and mouse (i) samples considering the mean difference in normalised expression values between cells and EVs (on a relative scale per row/miRNA). MiRNAs with read count ≥ 3 were considered in the correlation and clustering analysis. * miR 451a; + miR-144; x miR-6087; o miR-6240 ; □ miR-6239. a H E K b R D 4 c C 2 C 1 2 6 6 1 0 6 1 0 1 0 2 2 2 R = 0 .0 6 4 4 R = 0 .1 1 8 0 R = 0 .0 1 6 2 5 5 1 0 5 1 0 1 0 d d d 4 4 a 4 a a 1 0 1 0 1 0 e e s s e s r r r V V V 3 3 3 d d 1 0 1 0 d 1 0 E E E e e e , , , t t s s t s 2 2 i 2 i i 1 0 1 0 l l n 1 0 n l n a a u u a u 1 1 o 1 o m m o m 1 0 1 0 1 0 r r c c r c o o o 0 0 0 N N 1 0 1 0 N 1 0 n d n d n d 0 1 2 3 4 5 6 0 1 2 3 4 5 6 n d 1 0 0 1 0 1 1 0 2 1 0 3 1 0 4 1 0 5 1 0 6 n d 1 0 1 0 1 0 1 0 1 0 1 0 1 0 n d 1 0 1 0 1 0 1 0 1 0 1 0 1 0 N o rm a lis e d re a d N o rm a lis e d re a d N o rm a lis e d re a d c o u n t, c e lls c o u n t, c e lls c o u n t, c e lls d N e u r o 2 a e C 1 7 .2 1 0 6 1 0 6 2 2 R = 0 .0 9 1 3 R = 0 .5 4 9 1 1 0 5 1 0 5 d d 4 4 a 1 0 a 1 0 e e s s r r V 3 V 3 d 1 0 d 1 0 E E e e , , t t s 2 s 2 i 1 0 i 1 0 l l n n a a u u 1 1 o o m 1 0 m 1 0 r r c c o 0 o 0 N 1 0 N 1 0 n d n d n d 1 0 0 1 0 1 1 0 2 1 0 3 1 0 4 1 0 5 1 0 6 n d 1 0 0 1 0 1 1 0 2 1 0 3 1 0 4 1 0 5 1 0 6 N o rm a lis e d re a d N o rm a lis e d re a d c o u n t, c e lls c o u n t, c e lls f g row max row min row max row min Supplementary Figure S4.