Nature research article: The molecular evolution of spermatogenesis across mammals
of prechilled lysis buffer , 0.2 U µlfor 1 min at 4 °C. The supernatant was transferred to a new reaction tube and centrifuged at 500for 5 min at 4 °C. The supernatant was removed and the pellet resuspended in 0.67 vol. of freshly made fixation solution and incubated for 30 min at room temperature. The fixation was quenched by addition of Tris-HCl to a final concentration of 20 mM. The fixed nuclei were pelleted at 500for 5 min at 4 °C.
For platypus and chicken, a similar preparation method was used, but the nuclei were not fixed, given that this protocol gave optimal results for these species . In brief, tissue pieces weighing roughly 5 mg were homogenized in 100–150 µl mgfor 1 min at 4 °C. The supernatant was transferred to a new reaction tube and centrifuged at 500for 5 min at 4 °C. The supernatant was removed and the pellet resuspended in 0.67 vol. of Wash Buffer.
For bulk RNA-seq data generation, RNA was extracted using the RNeasy Micro kit . The tissues were homogenized in RLT buffer supplemented with 40 mM DTT. The RNA-seq libraries were constructed using the TruSeq Stranded messenger RNA LT Sample Prep Kit as described in ref.
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