Absolute blue sybr green rox

cDNA was prepared by first strand synthesis using the TURBO DNA-free Kit (REF AM1907; Invitrogen) and Superscript III (REF 5675; Invitrogen). First, 2.5 µg of total RNA was treated with DNase I and incubated at 37°C for 30 min. After inactivation with DNase Inactivation Reagent, 2 μl of DNase-treated RNA was combined with 4.5 μl of a master mix containing random hexamers Roche and dNTPs. Samples were incubated at 65°C for 5 min, then placed on ice. While on ice, 3.5 μl of RT master mix (1X first-strand buffer, DTT, RNaseOUT, SSIII [REF 56575; Invitrogen]) was added to each tube, then samples were incubated at 25°C for 5 min, 50°C for 1 h, and 70°C for 15 min. For RT-qPCR, 5.2 μl Absolute Blue SYBR Green ROX (REF AB-4162; Thermo Fisher Scientific) and 4.8 μl cDNA (diluted 1:20 prior to use) was added to each well of qPCR plates (REF 4346906; Life Technologies). Samples were run on a StepOnePlus RT-qPCR machine (Applied Biosystems). Average C_T values of triplicate reactions were used to calculate expression changes. RT-qPCR data was normalized to ACT1 for mitotic experiments and PFY1 for meiotic experiments (primers listed in Table S3).