GSM9759239: Spiderbgal-high cells from mouse acylic ovaries; Mus musculus; RNA-Seq
Ovary samples were collected separately from each individual at different days. Two ovaries were isolated and immersed in chilled M2 medium (Sigma, M7167). Surrounding fat and connective tissue were trimmed and removed. Two intact ovaries were minced in 1.5 ml Eppendorf tube and treated by enzymatic digestion in dissociation buffer (M2 medium supplemented with 30 ug/ml Liberase DH (Roche, LIBDH-RO) and 1000U/mL DNase (Thermo Fisher)) at 37°C for 15 min with shaking on thermomixer (1000 rpm). The tube was briefly spun at 1000 rpm and the supernatant was collected in a 15 ml centrifuge tube. Next, the remaining tissues were cut and incubated in a dissociation buffer for 10 min with shaking. The cell suspension was pooled with the supernatant in a 15 mL tube and neutralized with M2 medium with 1% BSA. The pooled cell suspension was filtered with a 45-um cell strainer  and was centrifuged. Cell pellets were incubated with RBC Lysis Buffer (Thermo Fisher) to remove red blood cells and subsequently washed with 1%BSA in PBS and 10% FBS in EMEM. Pelleted cells were resuspended in 10% FBS in EMEM and further submitted to Columbia Genomic Core for scRNA-seq library construction. library was performed by Columbia Genome Center according to the manufacter's instructions (3' single cell, 10x Genomics).