Epcam pe

Pups were euthanized using isoflourane. The testes were removed using tweezers, placed in a 1.5ml centrifuge tube and chilled on ice. When all testes had been removed, each pair was placed in 1 ml of type IV collagenase (Invitrogen) in a ultra low-attachment 6-well plate (Corning). All extraneous tissue and the tunica were removed and the seminiferous tubules were teased apart. The samples were then incubated 37°C 15 minutes and centrifuged for 5 minutes at 278g. Testes were then resuspended in 500µl of 0.25% Trypsin (Life Technologies) and incubated for 5 minutes at 37°C. After the incubation period the testes were agitated gently into suspension by pipetting. 500ul DMEM/10% FBS was added and the samples were centrifuged for 5 minutes at 200g.
For the P2.5 timepoints GFP positive cells were sorted as with embryonic time points. To sort germ cells at P10.5, the cells were washed with 1mL FACS buffer and then resuspended in 500µL FACS buffer. Then cells were then incubated with 1:160 EPCAM PE (Biolegend 118205) and 1:250µL H2-K^q 647 (Biolegend 115106) on ice for 20 minutes in the dark, then centrifuged 5 minutes at 200g and resuspended in 500µL FACS buffer. DAPI was added (1:1000, Life Tech) and the cells were strained through BD FACS tubes (Corning) before analysis. SSC^lo EpCAM^hi H2-K^q− cells were sorted into Buffer RLT or ATL for RNA or DNA extraction respectively.

Isolation methods were adapted from Strand et al.^{20 (link)} Briefly, samples were minced to a paste in the digestion medium (25 U/mL Collagenase type I (Gibco, Thermo Fisher), 0.25 mg/mL Dispase (Gibco) in RPMI 1640 medium with 10% FBS). Using 5 mL per 200 mg tissue, samples were incubated overnight in 50 mL tubes at 37°C with shaking and were spun down at 200 g for 5 minutes. Samples were next incubated in TrypLE Express (Life Technologies, Thermo Fisher) for 5 minutes at 37°C before being neutralized in RPMI 1640 medium containing 10% FBS, and passed through 18G needles to 70 μm cell strainers. Samples were incubated in ACK (ammonium-chloride-potassium) lysis buffer for 5 minutes at RT before flooding with phosphate buffered saline (PBS) and spinning down. Single-cell suspension was incubated in Zombie Viability Dye (BioLegend) for 10 to 15 minutes and spun down. Antibody cocktail (CD45-FITC, EpCAM-PE, CD26-APC, and CD49f-BV421, 1:100 dilution; BioLegend) was added to cells followed by incubation at 4°C for 30 minutes in the dark. Cells were filtered and sorted on BD FACSARIA (BD Biosciences, San Jose, CA) for luminal (CD45−, EpCAM+, CD26+, and CD49f−) and basal (CD45−, EpCAM+, CD26−, and CD49f+) cells. Sorted cells were washed with PBS and immediately suspended in TRIzol Reagent (Thermo Fisher) for subsequent RNA and protein isolation according to manufacturer's protocols.