Savant speed vac plus sc210a

The concentration of the protein content was estimated by the absorbance reading at 280 nm (NanoDrop 2000, Thermo Fisher Scientific, Wilmington, DE, USA). The protein content of approximately 30 µg of EVs was reduced using dithiothreitol (10 mM final concentration, 3 h at 37 °C) and alkylated with iodoacetamide (25 mM final concentration, 30 min in the dark at room temperature). Extracted proteins were incubated with trypsin (Promega, Madison, WI, USA) 1:50 (w/w) for ~20 h at 37 °C and 45 min at 56 °C thermoblock (Eppendorf, Hamburg, Hamburg, Germany). The reaction was stopped by adding trifluoroacetic acid to a final concentration of 1% (v/v). The digested peptide mixture was desalted in reverse-phase homemade microcolumns with POROS R2 resin (Applied Biosystems, Waltham, MA, USA). Samples were dried completely in a vacuum centrifuge (Savant Speed Vac Plus SC210A; Thermo Fisher Scientific, Waltham, MA, USA) and resuspended with 1% formic acid. The concentration of the peptide mixture was estimated by the absorbance reading at 280 nm (NanoDrop 2000, Thermo Fisher Scientific, Wilmington, DE, USA). Samples were stored at −20 °C for MS analysis.

For duplicate measurement, 1–10 μg of extracted DNA, together with 5 μl of 50 μM 5-aza-2′-deoxycytidine-¹⁵N₄ (internal standard, Toronto Research Chemicals, Toronto, ON, Canada), was added to 10 μl of 20 mg/ml NaBH₄ solution. In case of RNA, 1–10 μg of extracted RNA, together with 5 μl of 50 μM 5-azacytidine-¹⁵N₄ (internal standard, Thermo Fisher Scientific), was added to 10 μl of 20 mg/ml NaBH₄ solution. The mixture was incubated at room temperature with agitation for 20 min, and neutralised with 1 μl 5 M HCl. Reduced DNA or RNA were digested with 1 h of incubation at 37 °C following the addition of 40 μl of Digest Mix, an aqueous solution containing 50 U/ml benzonase (Sigma Aldrich, St Louis, MO, USA), 60 mU/ml phosphodiesterase (Sigma Aldrich), 20 U/ml alkaline phosphatase (Sigma Aldrich), 20 mM Tris HCl pH 8, 100 mM NaCl and 20 mM MgCl₂ as previously described.^{38 (link)} Samples were dried under vacuum (Savant Speedvac Plus SC210A, Thermo Fisher Scientific) and resuspended in 50 μl of CE buffer (10 mM Tris HCl pH 8.0 and 0.5 mM EDTA in Milli-Q water, Merck Millipore, Bedford, MA, USA) for LC–MS analysis.