title: GSM8805691: Cy6C_02O (MS233_049); Macaca fascicularis; RNA-Seq
library_construction_protocol: Collected cy ovaries, COCs and cultured follicles were placed in handling medium [DMEM/F-12 with HEPES (GIBCO, 11330057) containing 10% FBS (GIBCO, 10437028), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (GIBCO, 15070063)].  For isolating small follicles (primordial ~ preantral follicles) from cy ovaries, the ovarian cortical pieces were cut into 1 mm pieces after removal of medullary tissues. Then the tissue pieces were digested by a mixed digestion medium [aMEM (GIBCO, 12571063), 0.04 mg/ml Liberase DH (Roche, 5401054001), 10 IU/ml DNase I (Sigma-Aldrich, DN25)] for 20-75 min on a shaker at 37℃.  The incubation was terminated by wash with handling medium, and small follicles were manually collected.  Isolated follicles and COCs were photographed, followed by the dissociation process of oocytes and Granulosa cell.  Follicles and COCs were individually placed in 30 ml Accutase (Sigma-Aldrich, A6964) drops on a dish warmed by 37℃ plate, and oocytes and Granulosa cell were dissociated by pipetting up and down in a pulled fine glass capillary (Sutter Instrument, B100-75-10) with 30-120 mm in diameter, depending on the oocyte size.  For each follicle, about 15 Granulosa cell in the immediate vicinity of the oocyte were collected because of low abundancy of mRNA in these cells.  For primordial follicles, 15 Granulosa cell may not be obtained from a single follicle, in which case two or three primordial follicle-derived Granulosa cell were added together to make one sample.  At last, zona pellucida of GV/MII oocytes were removed by treating with acidic Tyrode's solution (Sigma-Aldrich, T1788) to obtain naked oocytes without contamination of Granulosa cell.  The removal of surrounding Granulosa cell from oocytes was confirmed under stereomicroscope (Leica, S8 APO) and inverted microscope (Olympus, IX71).  Dissociated oocytes and Granulosa cell from follicle grown in vivo and in vitro were manually picked up using glass capillaries under a stereomicroscope, and were used for SC3-seq. The cDNA synthesis and amplification were performed as described previously (Kurimoto et al., 2006; Kurimoto et al., 2007; Nakamura et al., 2016; Nakamura et al., 2015). The quality of the amplified cDNA was measured by a SYBR real-time PCR method (CFX384 Touch Real-Time PCR Detection System, BIO-RAD; Power SYBR Green PCR Master Mix, Applied Biosystems, 4367659) targeting the PPIA, GAPDH, DPPA3, DDX4, FOXL2, GJA1, and IGFBP7 genes. The SC3-seq libraries were constructed as described previously (Ishikura et al., 2021; Mizuta et al., 2022; Nakamura et al., 2016; Nakamura et al., 2015; Okamoto et al., 2021), and the sequencing was performed on the Illumina NextSeq 500/550 plat-form with a NextSeq 500/550 high output kit v.2 (75 cycles) (FC-404-2005; Illumina).
identifiers.namespace: GEO
identifiers.id: GSM8805691_r1