GSM5857327: Hs_11w4d_MS285_210; Homo sapiens; RNA-Seq
Fetal ovaries collected from human fetus were carefully dissected out in chilled PBS.  Fetal ovaries at 8-14 wpf were minced with scissors in 0.05% trypsin/EDTA/PBS solution (GIBCO, 15400-054), then incubated for 15 min at 37℃ with gentle pipetting every 5 min.  Dissociation medium [DMEM (GIBCO, 10313021) containing 10% FBS, 2mM L-glutamine (GIBCO, 25030081), 1X penicillin/streptomycin (GIBCO, 15070063), and 100μg/mL DNaseI (MERCK, DN25)] was added for further dissociation.  For SC3-seq analysis, cells were picked under the inverted microscope (OLYMPUS, IX71) to exclude multiplet cells.   For dissociating human rOvaries, tissues were incubated with TryPLE Express (1X) (GIBCO, 12604021) for 5 min.  After adding dissociation medium, cells were processed for single-cell analyses. Dissociated germ cells were manually picked using glass capillaries under the stereo microscope (Leica, S8 APO).  Following cDNA synthesis and amplification methods were previously described (Nakamura et al., 2015). The cDNA synthesis was performed according to the single-cell 3 prime RNA-seq (SC3-seq) method described previously (Nakamura et al., 2015).  The cDNA libraries for Nextseq500 (Illumina) were constructed as described previously (Ishikura et al., 2016).The sequencing was performed on an Illumina Nextseq500/550 platform with a Nextseq500/550 Hiqh output kit (Illumina).