Small RNA sequencing of mouse retinal exosomes

The current study isolated exosomes from healthy and degenerating mouse retinas. Retina degeneration was induced using a photo-oxidative damage model, involving the exposure of wild type C57B6J mice to a continuous bright light. Isolated exosomes were initially treated with RNaseA (10 micrograms/ml, 30 mins, 37 degrees celsius) to remove extra-exosomal RNA contamination. RNA was extracted from retinal exosomes using the mirVana RNA extraction kit (ThermoFisher) following the manufacturer's instruction for small RNA enrichment. The amount of RNA was quantified using a 2100 Agilent Bioanalyser using a small RNA chip. Sequencing libraries were prepared using the Capture and Amplification by Tailing and Switching kit (Diagenode, Leige, Belgium) following the manufacturers protocol for enriching the library for miRNA. The libraries were pooled and multiplex sequenced using the Illumina NextSeq500 platform acquiring single-end, 50 base-pairs reads from a single sequencing lane.