Sterile pestle

Proteins from bone marrow samples were extracted as previously described (31 (link)). Tissue was lysed in 500 μl of ice‐cold lysis buffer (40 mM Tris–HCl pH 7.4, 1% Triton X‐100, 40 mM Beta‐glycero phosphate, 5% Glycerol, 100 mM NaCl, 1 mM EDTA, 50 mM NaF and protease and phosphatase inhibitors (PMSF (1 mM), aprotinin (0.019 TIU/ml), leupeptin (1 μg/ml), NaF (5 mM) and Na₃VO₄ (1 mM)) per 100 mg and then crushed using a sterile pestle (Axygen). Lysates were incubated for 30 min on ice and sonicated 30 s ON\OFF for 10 cycles with a Bioruptor (Diagenode). Insoluble material was removed by high‐speed centrifugation at 4°C. Protein extracts were subjected to western blot analysis using anti-FANCI (Bethyl, A301-254A) or anti-FANCD2 (Abcam, ab108928).

Measurements of total body glucose and lactate were performed on a pool of 5 larvae per genetic condition using a fluorescence-based enzymatic detection kit (Biovision Inc.) as described before [81 (link)]. Briefly, larvae were collected in 1.5 ml microcentrifuge tubes, the excess of water was removed and samples were frozen stored at -80°C until further analysis. Upon defrosting, 15 μl PBS per larvae was added and tissues were disrupted with a sterile pestle (Axygen), on ice. Samples were spun at 14,000 x g for 10 min, at 4°C. The supernatant was immediately used for metabolite measurements according to manufacturers’ protocol. Fluorescence readings (Ex/Em 535/590) were taken with the FLUOstar Omega instrument (BMG Labtech). Results were read from a linear regression curve based on the dilutions of the standard. Statistical analysis between indicated groups was performed using the unpaired t-test and considered significant at P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***).

DNA extraction was done using the DNeasy Plant Tissue Mini Kit (Qiagen) with the following modifications. Pyrosome tissue was ground with a sterile pestle (Axygen, Tewksbury, USA) for 3 min prior to extraction. Seawater and pyrosome samples were lysed by bead beating with 0.55 and 0.25 mm sterile glass beads at 30 Hz for 2 min after addition of lysis buffer, freeze-fractured three times, incubated with Proteinase K (VWR Chemicals, Solon, OH, USA) at 20 mg/mL for 1 h at 55 °C, and incubated with RNase A at 100 mg/mL for 10 min at 65 °C. To minimize amplification of eukaryotic host DNA, the primer pair 515F‐Y/806R was chosen to amplify the 16S rRNA V4 hypervariable region with conditions as published.^{34 (link)} Reactions were performed with 0.5–2 ng of DNA using the QuantaBio 5Prime HotMasterMix (Qiagen Beverly, MA, USA). The Agilent High Sensitivity Kit in the Bioanalyzer (Agilent Technologies, Waldbronn, Germany) confirmed amplicon size. Triplicate reactions from each sample were pooled and paired-end sequenced with Illumina MiSeq v.3 (Illumina, San Diego, USA).