Reverse transcription aid kit

Isolated cells were treated with Trizol reagent, mixed with 200 µl chloroform, and centrifuged at 8,000 g at 4°C. The supernatant was recovered, mixed with 500 µl 2-propanol, and centrifuged at 8,000 g at 4°C. The mRNA pellet was extracted with 75% ethanol to increase RNA purity. After centrifugation, ethanol was removed by evaporation, and the pellet was dissolved in diethylpyrocarbonate-treated distilled water (Tech & Innovation, Chuncheon, South Korea). A reverse-transcription aid kit (Thermo Scientific, MA, USA) was used to synthesize cDNA, which was then subjected to q-PCR using SYBR Green PCR Master Mix (Roche, Basel, Switzerland) and a LightCycler 480 Instrument II (Roche). GAPDH was used as an internal control. The sequences of all primers used for PCR are listed in Table 1.

Fresh isolated tissues from WT and Top3β^−/− mice were treated with Trizol reagent, mixed with 200 µL chloroform, and centrifuged at 4 °C for 15 min at 8000× g. The supernatant was recovered, mixed with 500 µL 2-propanol, and centrifuged at 4 °C for 10 min at 8000× g. The mRNA pellet was extracted with 75% ethanol to increase RNA purity. After centrifugation, ethanol was removed by evaporation and the pellet was dissolved in diethylpyrocarbonate-treated distilled water (Tech & Innovation, Chuncheon, South Korea). A reverse-transcription aid kit (Thermo Scientific, Foster City, CA, USA) was used to synthesize cDNA, which was then subjected to q-PCR using SYBR Green PCR Master Mix (Roche, Basel, Switzerland) and a LightCycler 480 Instrument II (Roche). GAPDH was used as an internal control. Sequences and sources of all primers used for PCR are listed in Supplementary Table S1 [16 (link),62 (link),63 (link),64 (link),65 (link)]. The Spo11 TaqMan probe was used.