Myone streptavidin dynabeads

MyOne Streptavidin dynabeads (Life Technologies) were blocked in BSA and yeast tRNA and were preloaded with 5′-biotinylated oligonucleotides or control (biotin-362as) oligonucleotides (Integrated DNA Technologies, Coralville, Iowa, USA). DNase pretreated RNA from HEK293T cells was added to the beads and incubated for 2 h at RT. The supernatant containing the poly(A)-depleted RNA fraction was collected and used to generate cDNA. The cDNA was then assessed by semi-quantitative reverse transcription PCR (semi-qRT-PCR).

sEVs were incubated with barcoded antibodies (5 nM of each) in 1× PBS buffer (Thermo Fisher Scientific; Cat. No. 18912014) in the presence of 50–500 nM of Z(WT), at 4°C overnight whilst rotating. MyOne Streptavidin Dynabeads (Thermo Fisher Scientific; Cat. No. 65306) were covered with biotinylated Cholera Toxin B subunit (CTB, Thermo Fisher Scientific; Cat. No. C‐34779). Approximately 20 × 10⁶ sEVs (as determined by NTA analyses) were incubated with circa 200 × 10⁶ CTB coated beads in PBS‐CTD buffer (1x PBS with 0.05% casein (w/v), 0.1% Tween‐20 (v/v), 1 mg/ml salmon sperm DNA (Thermo Fisher Scientific; Cat. No. 15632011) and 50–500 nM of Z(WT)), with rotation at 4°C for 10 minutes. The 1 sEV to 10 bead (0.1 vesicles per bead [VPB]) relationship was used to ensure that the majority of the sEV‐carrying beads captured a single vesicle. Three times washing with PBS‐CTD buffer was then applied and after the last wash, the beads were resuspended in 200 μl 1× PBS buffer. A fraction of the beads was used in emulsion PCR (emPCR) and another for quantitative PCR (qPCR). qPCR samples were accompanied with negative (antibody‐background) controls for which the whole above‐process was carried out without inclusion of sEVs in the initial step.

The emulsion breakage and library preparation steps were done as previously described by Redin et al. (2019 (link)), except for modifications in the PEG‐based purification. Briefly, following aqueous phase recovery and PCR product purification by 20% PEG (Sigma Aldrich), biotinylated amplicons were enriched using MyOne Streptavidin Dynabeads (Thermo Fisher Scientific). After NaOH treatment, an indexing reaction was performed using a master mix containing Phusion Hot Start Flex (New England BioLabs; Cat. No. M0536L) and 0.2 μM of i5‐H1 and i7‐H4’ indexing primers (see Table S2 for sequences) using the following program: two cycles of 2 min at 95°C, 1 min at 60°C (with 20% ramp speed) and 10 min at 72°C (with 3% ramp speed). The final product was then purified three times using 16% PEG and the quality was examined by Bioanalyzer (Agilent). The libraries were quantified by Qubit fluorometer (Invitrogen) and sequenced using Reagent Kit v3 (150‐cycle) for the MiSeq instrument (Illumina).