2 butanol

The solvents n-hexane, 2-propanol (iPrOH), formic acid (FA), acetonitrile (ACN), and methanol (MeOH) of ultrahigh-performance LC/MS grade were purchased from Biosolve (Valkenswaard, Netherlands). Water was produced in-house with a Barnstead GenPure System from Thermo Scientific (Waltham, MA, USA). Methyl tert-butyl ether (MTBE) of pro analysis quality was obtained from Carl Roth (Karlsruhe, Germany). 2-Butanol of extra-pure quality was purchased from Thermo Fisher Scientific (Waltham, MA, USA). 2,6-Di-tert-butyl-4-methylphenol (BHT) of gas chromatography quality and EDTA of ACS reagent grade were purchased from Sigma-Aldrich (St Louis, MO, USA). Ethyl acetate and chloroform (CHCl₃) of LC grade, zinc sulfate heptahydrate of pro analysis quality, and phosphate-buffered saline (PBS) of liquid, sterile-filtered, suitable-for-cell-culture grade were obtained from Merck Chemicals (Darmstadt, Germany). Tris (hydroxymethyl)aminomethane–HCl buffer was obtained from Bioanalytic (Umkirch, Germany). Unlabeled and deuterium-labeled PUFA and eicosanoid standards were purchased from Cayman Chemicals (Ann Arbor, MI, USA); the detailed information can be found in Table S1.

After PCR the emulsions were transferred to a 50 mL Falcon tube. 125 μL of 2-butanol (Thermo Fisher Scientific) was added to each well that had contained emulsion, and the butanol was then transferred to the same 50 mL tube. The tube was vortexed for 30 sec rigorously and then centrifuged at 3000 x g for 6 min. A magnet was used to retain the pellet of aptamer particles while removing the supernatant. 1.2 mL of breaking buffer (100 mM NaCl, 1% Triton X-100, 10 mM Tris-HCl, pH 7.5, and 1 mM EDTA) was added to the particles, and the mixture was transferred to a new 1.5 mL tube. The 1.5 mL tube was vortexed and centrifuged at 21,000 x g for 1 min. Using a magnetic rack, the supernatant was removed with a 1 mL micropipette. Another 1 mL of breaking buffer was added to the particles and removed as described above for multiple cycles until absolutely no white film was visible on the top of the supernatant. 400 μL of breaking buffer was then added, and the sample was transferred to a new 1.5 mL tube. Once again 1 mL of breaking buffer was added, vortexed, and centrifuged at 21,000 x g for 1 min. The supernatant was removed, and the aptamer particles were resuspended in 1 mL of wash buffer.

After PCR, the emulsions were transferred to a 50 mL Falcon tube. 125 μL of 2-butanol (Thermo Fisher Scientific) was added to each well to wash residual emulsion, and the butanol was then transferred to the same 50 mL tube. The tube was vortexed for 30 seconds and then centrifuged at 3000 × g for 5 minutes. The supernatant was removed while retaining the pellet of aptamer particles at the bottom of the tube. 1.2 mL of breaking buffer (100 mM NaCl, 1% Triton X-100, 10 mM Tris-HCl, pH 7.5, and 1 mM EDTA) was added to resuspend the particles, and the mixture was transferred to a new 1.5 mL tube. The 1.5 mL tube was vortexed and centrifuged at 21,000 x g for 1 minute. Using a magnetic rack, the supernatant was removed with a 1 mL micropipette. Another 1 mL of breaking buffer was added to the particles, which were then transferred to a new tube, and the supernatant was removed as described above for multiple cycles until no residual oil (white film) was visible on the top layer. On the last wash, 150 μL of breaking buffer was added to the sample, which was then transferred to a new 1.5 mL tube. The supernatant was removed, and the aptamer particles were washed three times and resuspended with 500 μL SELEX buffer.