RNA-seq of the RNA cargo of extracellular vesicles released in perfusate and bile during ex situ isolated liver perfusion of porcine livers exposed to 60-minutes warm ischemia vs. control (no warm ischemia)
Eight mL of perfusate per time point (1, 3, 6h of liver normothermic machine perfusion) were immediately centrifuged at 1000 g for 10 min at room temperature. The supernatant was aliquoted in 1 mL microtubes, supplemented with 10 microL thrombin (Merck, Sigma-Aldrich) and centrifuged at 10000 g at room temperature for 10-minutes. The supernatant was then transferred in another microtube and centrifuged at 16500 g at 4°C for 20-minutes. The resulting supernatant was filtered through a 0.2micron PES filter before EVs separation.EVs were separated with charge-based precipitation with protamine (Merck, Sigma-Aldrich) and polyethylene glycol 35000 (Merck, Darmstadt, Germany) (P/PEG).Briefly, perfusate samples ready for precipitation were supplemented with P/PEG (1 volume P/PEG – 4 volumes sample) and incubated overnight at 4°C while gently rotating. The mixture was then centrifuged at 1500 g at room temperature for 30-minutes, the supernatant discarded, and the final pellet was resuspended in the appropriate buffer. The miRNeasy Micro kit from Qiagen was used to extract total RNA from EVs following manufacturer's instructions. RNA purity and concentration were assessed from 1 mL of sample with NanoDrop and expressed as ng/mcL. Sequence libraries were prepared with the small RNA Lexogen  Library prep kit according to the manufacturer's protocol. Samples were indexed to allow for multiplexing. Libraries were sequenced on a Illumina HiSeq4000 platform. Single-end reads of 50bp length were produced with a minimum of 1M reads per sample.