Bravo pipetting robot

cDNA amplifications were carried out in a total volume of 13 μL containing 6 ng of cDNA templates, 6.5 μL of SYBR Green (ThermoFisher Scientific, Saint-Aubin, France), and 300 nM of each primer. Specific primers are listed in Table S6. Samples were dispensed using the Bravo pipetting robot (Agilent Technologies, Les Ulis, France). Reactions were performed in 384-well plates (Life Technologies) in a QuantStudio 12K Flex system. The amplification condition was 20 s at 95 °C followed by 40 cycles (1 s at 95 °C, 20 s at 60 °C) and a final step of denaturation of 15 s at 95 °C, 1 min at 60 °C, and 15 s at 95 °C. Melting curves were also obtained to ensure the specificity of PCR amplifications. The size of the amplicons was verified by agarose gel electrophoresis (E-gel 4%, Life Technologies). The relative expression level of each gene was normalized to two housekeeping genes, as recommended by the MIQE guidelines [192 (link)]: Gapdh and actin β (Actb), which were identified and validated as the most stable and suitable genes for RT-qPCR analysis in rodent testis development [193 (link)]. Data were analysed using the 2^−ΔΔCt method [194 (link)]. The Kruskall–Wallis test was used to determine significant differences.

Peptides were prepared semi-automated on a Bravo pipetting robot (Agilent), similarly to as described previously¹¹ . During each incubation step, plates were tightly sealed with two stacked aluminum lids to avoid evaporation (Thermo Fisher Scientific, AB0626). For this, plates were removed from the freezer and centrifuged. The wells were then washed on the robot with 28 µl of 100% ACN and dried in a SpeedVac (Eppendorf) at 45 °C for 20 min. Shapes were then resuspended in 6 µl of 60 mM triethylammonium bicarbonate buffer (pH 8.5, Sigma) with 0.013% DDM (Sigma), and cooked for 30 min at 95 °C in a PCR cycler at a lid temperature of 110 °C. After addition of 1 µl of 80% ACN (final concentration 10%), samples were incubated for another 30 min at 75 °C, cooled briefly, and 1 µl with 4 ng LysC and 6 ng trypsin was added. The samples were digested for 18 h, and then 1 µl of either intermediate (CD₂O) or heavy formaldehyde (¹³CD₂O) was added to a final concentration of 0.15%. Without delay, either light (NaBH₃CN) or heavy (NaBD₃CN) sodium cyanoborohydrate were added to 0.023 M to retrieve Δ4 and Δ8 dimethyl-labeled single-shape samples. The sealed plate was then incubated at room temperature for 1 h, and the reaction was quenched to 0.13% ammonia and acidified to 1% TFA.