Precellys evolution homogenizer

Enzymatic cycling assays to determine NADP(H) and NAD(H) levels were performed according to the NADP⁺/NADPH Quantification Kit (Sigma) and NAD⁺/NADH Quantification Kit (Sigma). Cells were grown in Delft + 100 mg L⁻¹ histidine + 100 mg L⁻¹ uracil medium, then 5 OD_600nm of cells were collected at later exponential (EX) phase (OD_600nm ≈ 2) and post-diauxic (PD) phases. Collected cells were immediately added to 20 ml of cold methanol (pre-chilled to −80 °C) to quench cellular metabolism. Cells were centrifuged at −10 °C for 4 min (4000×g), then supernatant was removed. Cell pellets were freeze-dried for 2 h and stored at −80 °C until measurement. The dried cell pellet was resuspended in 500 µl of extraction buffer (Sigma) and added to 0.2 g of prechilled glass beads (425–600 µm diameter, Sigma). Cells were lysed via four rounds of vortexing for 20 s at speed 6200 rpm on the Precellys Evolution Homogenizer (Bertin technologies) followed by incubation for 1 min on ice. The cell lysates were centrifuged at 14000×g for 1 min at 0 °C, and supernatant was used to measure NADH(H) and NAD(H) amounts following the manufacturer’s instructions (Sigma).

Samples were thawed to room temperature and 2-mercaptoethanol (Sigma-Aldrich, St. Louis, Missouri, USA) was added to each sample to a final 1% concentration and mechanically homogenized 2 × 50 s for 6500 RPM using Precellys Evolution homogenizer (Bertin Technologies SAS, France). The samples (and the DNA extraction negative control) were then processed for DNA extraction using the AllPrep DNA/RNA 96 Kit (Qiagen Hilden, Germany) according to the manufacturer’s spin protocol and the eluted DNA was stored at − 20 °C until further analysis. The ZymoBIOMICS Fecal Reference standard (Zymo Research, USA) was processed for DNA extraction using the ZymoBIOMICS DNA Miniprep Kit (Zymo Research, USA) according to the manufacturer’s spin protocol and the eluted DNA was stored at − 20 °C until further analysis.

DNA was extracted from up to 25 mg of frozen tissue using a DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany). Prior to extraction the tissue was homogenized in a Precellys Evolution homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France) with 180 µL lysis buffer using 2.8 mm zirconium oxide beads in reinforced 2 mL microtubes. DNA was then extracted according to the manufacturer’s instructions.

For tissue collection, female rainbow trout were euthanized using a lethal dose of tricaine methanesulfonate (MS222) at a concentration of 200 mg/L. Ovaries were dissected and ovarian samples were either frozen in liquid nitrogen and stored at − 80 °C until RNA extraction or homogenized in RIPA buffer (NaCl 150 mM, deoxycholic acid 0.5%, NP40 1%, SDS 0.1%, Tris 50 mM, pH 8) using a Precellys Evolution Homogenizer (Ozyme, Bertin Technologies) to extract proteins. After centrifugation, protein concentration in the supernatant was determined using the Coomassie (Bradford) Protein assay Kit (Thermo Fisher Scientific, Rockford, IL, USA) and bovine serum albumin (BSA) as a calibration standard. Supernatants were then stored at − 20 °C until use.

Four aliquots of 5 g were obtained from each defrosted stool sample. Each aliquot was transferred in a 15 ml tube containing a mix of zirconia beads (25 of 2.0 mm diameter and 3 of 5.0 mm diameter).
Five milliliters of buffer ASL (QIAamp DNA Stool Mini Kit, Qiagen, Hilden, Germany) were added to each tube. Then, all tubes were subjected to a grinding process, using Precellys Evolution Homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France).
A set of 3 cycles of 30 s each, at 9,600 rpm, was carried out, with a resting time of 1 min at room temperature between each cycle. The resulting homogenate, together with the beads, was transferred into four 50 ml tubes and a further 40 ml of buffer ASL was added to each of them.
The samples were then vortexed for 10 s, incubated at room temperature for 4 min, vortexed for 10 s, and finally centrifuged for 3 min at 4,500 rpm.
Subsequently, the supernatant was transferred into a new 50 ml tube and vortexed for 10 s. Then, 1.2 ml of the supernatant were taken from each of the four 50 ml tubes and transferred into four 1.5 ml eppendorf tubes (for DNA extraction and purification).