By computational gene regulation inference analysis, we identified sex-specific, sequential waves of master regulator genes during germ cells differentiation and unveiled that the meiotic initiator Stra8 is regulated by positive and negative master regulators acting in an antagonistic fashion. Here, we describe a comprehensive characterization of gene expression dynamics during sex determination based on single-cell RNA sequencing on 14,750 XX and XY mouse germ cells between embryonic days 10.5 and 16.5. Single-cell transcriptomics reveals temporal dynamics of critical regulators of germ cell fate during mouse sex determinationĮxpression profiling by high throughput sequencingĭespite the importance of germ cell differentiation for sexual reproduction, gene networks underlying their fate remain unclear. Wash suspension several times by centrifugation in the balanced salt solution.GEO help: Mouse over screen elements for information.Fresh dispase can be added to the fragments if further disaggregation is required. Filter the cell suspension through a sterile, stainless steel or nylon mesh to separate the dispersed cells and tissue fragments from the larger pieces.Incubate at 37☌ for 20 min to several hours.Add dispase (0.6 to 2.4 U/ml in calcium and magnesium-free balanced salt solution).Wash the tissue pieces several times in a calcium and magnesium-free balanced salt solution. Mince tissue into 3 to 4 mm pieces with a sterile scalpel or scissors.Resuspend the pellet in culture medium.Wash suspension several times by centrifugation in HBSS.Fresh collagenase can be added to the fragments if further disaggregation is required. Filter the cell suspension through a sterile stainless steel or nylon mesh to separate the dispersed cells and tissue fragments from the larger pieces.Addition of 3 mM CaCl 2 increases the efficiency of dissociation. Add collagenase (50 to 200 U/ml in HBSS).Wash the tissue pieces several times with Hanks' Balanced Salt Solution (HBSS). Filter the cell suspension through sterile, stainless steel mesh (100 to 200 µM) to completely disperse any remaining tissue.
If using a serum-free medium, also add soybean trypsin inhibitor. Add warm, complete media to the tissue pieces and gently disperse the tissue by pipetting.Incubate the tissue pieces with residual trypsin at 37☌ for 20 to 30 min. Decant and discard the trypsin from the tissue pieces.Incubate at 4☌ for 6 to 18 h to maximize penetration of the enzyme with little trypsin activity.Add 0.25% trypsin in a balanced salt solution without calcium or magnesium (1 ml of trypsin for every 100 mg of tissue). Place the container with the tissue pieces on ice, and remove any remaining supernate.Allow the tissue pieces to settle, and remove the supernate. Wash the tissue pieces by resuspending in a balanced salt solution without calcium and magnesium. After dissecting off unusable tissue, mince the remaining tissue into 3 to 4 mm pieces with a sterile scalpel or scissors.Discard supernatant and suspend cell pellet with 2 to 5 ml of fresh growth medium. Centrifuge for 5 to 10 minutes at 100 × g.Dilute in 2 to 5 ml of cell culture growth media and transfer cell suspension to a 15 ml centrifuge tube.Incubate at 37☌ until cells have detached (observe at 5 minute intervals).Rock vessel to coat cell sheet completely. 2 ml in a 75 cm 2 flask) of prewarmed TrypLE to flask. Rinse flask with 5 ml of Dulbecco’s Phosphate Buffered Saline (D-PBS) without calcium and without magnesium (GIBCO cat. Optimal conditions and concentrations employed for individual systems should be determined empirically. The following general procedure can be used to remove various cell lines from cultureware while maintaining cellular integrity. TrypLE products are formulated to allow direct substitution into your existing protocols.
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