Mytilus edulis

Three times volume TE was added to each sample, RNaseA (0.2 μg/ml final concentration, Qiagen) was added and samples were incubated at 37 °C for 30 min to degrade RNA. To degrade protein, proteinase K (Sigma-Aldrich) (0.2 μg/ml final concentration) was added and incubated at 55 °C for 1 h.
To isolate DNA, 400 μl phenol:choloform:isoamylalcohol (Ambion) was added to each sample and phases separated using 2 ml Heavy Phaselock (Qiagen) according to manufacturer’s instructions. The aqueous layer was recovered and NaCl (200 mM final concentration) and 30 μg glycogen from Mytilus edulis (Sigma-Aldrich) was added. 800 μl ethanol was added and the samples were incubated for 1 h at −80 °C prior to centrifugation at 13,300 rpm at 4 °C for 10 min. Supernatant was removed, and the pellet washed with 75 % Ethanol, before being air-dried and resuspended in 20ul 10 mM Tris–HCl pH 8.5. DNA concentration was measured using the QuBit 2.0 system (Invitrogen).

Total cellular RNA, including miRNA, was isolated with the miRNeasy Mini Kit (Qiagen) following the manufacturer’s protocol. All optional washing steps were included and RNA was eluted in a final volume of 30 μl RNase-free water. Total exosomal RNA, including miRNA, was isolated using the protocol described for cells with slight modifications: Exosome samples were pre-treated with 100 ng/μl RNAse A (Roche) for 30 min at 37°C, immediately before extracting RNA. Prior to the addition of chloroform and phase separation, 12 μg glycogen from Mytilus edulis (Sigma) were added to the sample. RNA concentrations were measured with the NanoDrop ND-1000 at 260 nm. RNA quality was assessed with the Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany) using the Agilent RNA 6000 Pico Kit (total RNA) and the Agilent Small RNA Kit (small RNA). The 2100 Bioanalyzer Expert Software B.02.08. (Agilent) was applied to generate electropherograms. RNA samples were stored at -80°C until further analysis.