Humic acid solution

The GRZ (GRZ-AD) strain of N. furzeri³ was gifted by Prof. Adam Antebi. Fish were maintained at 26.5 °C, 0.7 conductivity on 12/12 h light/dark cycle in the fish breeding system (MEITO, Nagoya, Japan) at a density of 1 fish per 1.4 L tank for adult fish. Fish were fed freshly hatched brine shrimp twice a day from Monday to Saturday, and once a day on Sunday, fish that were over 2 weeks old were also fed bloodworms (Kyorin, Himeji, Japan). For mating, one adult male and 3–4 female fish were kept in 4 L tanks, and females spawned on a sand substrate in the plastic cases. Embryos were collected and incubated in the egg water (0.03% sea salt containing methylene blue). Ten days later, the embryos were plated on sterile dry peat moss until they were ready to hatch. Usually, one month after egg collection, embryos were ready to hatch and incubated in the 0.07% ice-cold humic acid solution (53680, Sigma-Aldrich, St. Louis, MO, USA) for 30–60 min, and transferred into the 4 L hatching tank with air supply. Hatched fry were kept in a hatching tank, and half of the solution was changed with the fish breeding system water every day. After 2 weeks, by which time they had grown to be around 1–1.5 cm, they were transferred to 1.4 L tanks similar to the adult fish.

Humic Acid-inhibited extracts were generated by combining 50 μL aliquots of each whole blood dilution with 50 μL Buffer ATL, 10 μL Proteinase K, and 100 μL Buffer AL (containing cRNA) [43 ]. These solutions were vortexed, incubated at 50^∘C for 10 min, then briefly centrifuged. Three volumes (15, 22, and 35 μL) of 2 mg/mL humic acid solution (Sigma Aldrich) were added to the cell lysate solutions which were subsequently incubated at room temperature for two hours, vortexing every 30 min to mix. After incubation, the extraction protocol was resumed to completion.