Isolation and Characterization of Mouse Germ Cells

Mouse cDNA was prepared from various tissues of adult mice, or from 5- to 42-day-old mouse testes. Alternatively, cDNA was prepared from spermatocytes and spermatids, which were isolated and purified from mouse testes as previously described^{43 (link)}. Briefly, testes from 24-day-old mice were dissected to remove the tunica albuginea. The decapsulated testes were treated with collagenase B (Roche, Tokyo, Japan) and DNase I (Sigma-Aldrich, St. Louis, MO), followed by treatment with trypsin (ThermoFisher, Waltham, MA) and DNase I. The isolated germ cells were then washed with phosphate-buffered saline (PBS) and stained with Hoechst 33342 in 5% bovine serum albumin (BSA) in PBS at 34°C for 1 hour. Spermatocytes and spermatids were sorted based on DNA content and light-scattering properties by BD FACSAria™ IIu (BD Biosciences, Tokyo, Japan). Reverse transcription quantitative PCR (RT-qPCR) was performed using the THUNDERBIRD™ Probe and SYBR^® qPCR Mix (TOYOBO, Osaka, Japan) and the CFX384 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA). The sequences of primers are listed in Table S2.