Advanced dmem f12 glutamax

A total of 31 adult ovarian tissue samples (2–8 mm diameter) containing (whole or parts of) a single visible follicle (1–2 mm or 2–5 mm diameter) or without visible follicles were dissociated for single cell transcriptomics as previously described^{45 (link)}. Briefly, individual tissue samples from adult ovary were incubated overnight on ice with 1 mg/ml Collagenase Type II (Life Technologies) in 0.25% Trypsin-EDTA (Life Technologies). Next, the samples were centrifuged at 160 × g for 3 min and incubated with Advanced DMEM/F12+ Glutamax (Life Technologies), 1x Insulin-Transferin-Selenium (Life Technologies), 1x Penicillin/Streptomycin (Life Technologies) and 27 IU/ml RNase-free DNase I (Qiagen) at 37 °C for 30 min to 2 h. The digestion was stopped by adding 10% of fetal calf serum (Gibco), followed by a filtration step through a 100 µm strainer (Corning). Samples were centrifuged at 160 × g for 5 min and stored in liquid nitrogen in Bambanker (Nippon Genetics).

The ovarian tissue pieces were dissociated for single-cell RNA sequencing. In brief, the tissue pieces frozen in cryopreservation medium were thawed in a water bash at 37 °C, washed twice with cooled RPMI 1640 medium containing 0.04% bovine serum albumin (BSA), and further minced into approximately 0.5 mm³ pieces in RPMI 1640 medium containing 0.04% BSA. The pieces were subjected to digestion with enzymes, 1 mg/mL collagenase Type II (Life Technologies, Grand Island, NY, USA) and 0.25% trypsin-EDTA (Life Technologies, USA) on ice overnight. The digestion process was stopped by adding DMEM supplemented with 10% of fetal calf serum (Gibco, Paisley, UK). To collect the dissociated cells, the cell suspension was centrifuged at 160× g for 3 min. Next, the dissociated cells were incubated with advanced DMEM/F12 Glutamax (Life Technologies) containing 1% insulin-transferrin-selenium (Life Technologies, USA), 1% penicillin-streptomycin (Life Technologies, USA), and 27 IU/mL RNase-free DNase I (Qiagen, Hilden, Germany) at 37 °C for 1 h. The cells were resuspended in DPBS containing 2% FBS and passed through a 40 μm cell strainer (Corning, NY, USA) to remove remaining cell aggregates.