Proximity barcoding assay (PBA) processing
Surface protein profiles of individual extracellular vesicles (EVs) were obtained using the PBA (ExoSeek® panel260, Secretech, Shenzhen, China), following the procedure outlined in the PBA method paper (Wu et al., 2019). In brief, EVs were first incubated with a mixture containing 260 antibodies. The antibody panel included general EV markers such as CD9, CD63, and CD81, along with biomarkers reported in the literature as potential contributors to cataract formation and mostly cellular adhesion molecules that are critical for EV targeting, cellular uptake, and signaling. Each antibody was conjugated with oligonucleotides containing unique protein tags for antibody identification, molecular tags for protein quantification, and a universal sequence for primer binding. Subsequently, the EV-antibody complexes were captured in microplate wells coated with cholera toxin subunit B (CTB), which binds to GM1 sphingolipid on the EV membrane. These complexes were then detected by individual rolling circle amplification (RCA) products, which contained DNA sequences complementary to those conjugated to the antibodies, serving as barcoding templates. All proteins from a single EV shared the same barcode. The unique protein and molecular tags, along with the RCA products (referred to as ComplexTag), were linked by annealing and PCR amplified for detection.
The libraries were sequenced on the DNBSEQ-T7 platform (MGI, Shenzhen, China) or the NovaSeq S4 system (Illumina, USA) using paired-end 150 sequencing. Raw sequencing data in bcl format were converted to FASTQ files using bcl2fastq software for downstream analysis.